Secreted Factors from Human Umbilical Cord Blood Cells Protect Oligodendrocytes from Ischemic Insult

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Graduate Theses and Dissertations Graduate School

2011

Derrick Rowe University of South Florida, [email protected]

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Secreted Factors from Human Umbilical Cord Blood Cells Protect

Oligodendrocytes from Ischemic Insult

Derrick D. Rowe

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Pharmacology and Physiology College of Medicine University of South Florida

Major Professor: Keith R. Pennypacker, Ph.D. Craig Doupnik, Ph.D. Marcia Gordon, Ph.D. Samuel Saporta, Ph.D. Lynn Wecker, Ph.D. Alison Willing, Ph.D.

Date of Approval: June 27, 2011

Keywords: Oxidative stress, Hypoxia, Peroxiredoxin 4, Stroke, Metallothionein 3, Leukemia inhibitory factor

DEDICATION

I dedicate this dissertation to my family and friends who have supported me throughout the years both financially and emotionally and have always been there to keep me grounded and focus at the task at hand until completion. I could not have done it without you.

I would like to thank my parents for their continuing support and for always being there when I’m in need. I would like to offer special thanks to the crew,

Toni, Timi and Chante for grounding me whenever I was flying too high and for always lifting me up whenever I was knocked down and out. Thanks guys, I’m forever in debt to you.

ACKNOWLEDGMENTS

I would never have been able to finish my dissertation without the

guidance of my major professor Dr. Keith Pennypacker, my committee members

and the support of friends and my wonderful family.

I would like to personally and wholeheartedly thank my major professor,

Dr. Keith Pennypacker for his excellent guidance, patience, and providing me

with an excellent research atmosphere. I would like to thank Franjesca Bailey

Jackson for her guidance and push to apply for the McKnight Doctoral Fellowship

which supported my Research. I would like to personally thank the McKnight

family for their continuing support.

I would like to thank the faculty and staff at the University of South Florida.

I’d like to thank the members of my laboratory former and present Dr. Ted Ajmo,

Dr. Aaron Hall, Dr. Christopher Leonardo, Dr. Glenn Roma, Jesus Recio, Lisa

Collier and Hilary Seifert who aided in the completion of my dissertation.

Finally I would also like to thank my parents, and sisters.

TABLE OF CONTENTS

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

ABSTRACT ...... ix

CHAPTER 1: INTRODUCTION ...... 1 Overview of Stroke...... 2 Types of Stroke ...... 3 Ischemic Stroke...... 3 Hemorrhagic Stroke ...... 4 Ischemic Stroke Pathology ...... 4 Energy Depletion...... 4 Immune Response ...... 5 Reperfusion ...... 6 Animal Models of Stroke ...... 6 Global Ischemia ...... 7 Focal Ischemia ...... 8 Moving Past the Neuron Centric Perspective ...... 10 White Matter ...... 11 Overview ...... 11 OL Development ...... 11 Stroke and Oligodendrocytes ...... 14 Oxidative Stress ...... 14 Clinical Trials ...... 17 Neuroprotective Agents ...... 18 Addressing Failure ...... 19 Antithrombotics ...... 20 Tissue Plasminogen Activator ...... 20 Stem Cells ...... 21 Human Umbilical Cord Blood Cell ...... 23 Origin ...... 23 Cellular Protection ...... 24 Anti-inflammatory Effects ...... 25 HUCB cells Secrete Soluble Factors ...... 26 Leukemia Inhibitory Factor ...... 27 Akt Survival Pathway ...... 30

Antioxidant Expression in the Brian ...... 31 MT3 ...... 31 Prdx4 ...... 33 HUCB Cells Therapy to Protect the White Matter OL ...... 35 References ...... 35

CHAPTER 2: CORD BLOOD ADMINISTRATION INDUCES OLIGODENDROCYTE SURVIVAL THROUGH ALTERATIONS IN GENE EXPRESSION ...... 51 Abstract ...... 52 Keywords ...... 53 Abbreviations ...... 53 Introduction ...... 54 Results ...... 56 Characterization of Mature OLs ...... 56 Secreted Factors from HUCB Cells Protect Mature OLs ...... 56 HUCB Cell Protection is Associated with Changes in Gene Expression ...... 57 qRT-PCR Verification of Microarray Results ...... 57 HUCB Cells Reduce Infarct Volume ...... 58 HUCB Cells Protect OLs In Vivo ...... 59 HUCB Cells Induce Protein Expression ...... 59 Protein Expression and Localization ...... 60 Comparison of Gene Promoter ...... 61 Discussion ...... 61 Experimental Procedure ...... 66 Animal Care ...... 66 Mixed Glial Culture Preparation ...... 67 OL Culture Purification ...... 67 Oxygen Glucose Deprivation...... 68 LDH Assay ...... 69 RNA Collection and Purification ...... 70 Gene Array ...... 70 Quantitative Real Time Polymerase Chain Reaction ...... 71 Determination of Promoter Response Elements ...... 72 Laser Doppler Blood Flow Measurement ...... 72 MCAO and HUCB Cell Treatment ...... 73 Fluoro-Jade Histochemistry...... 74 Immunohistochemistry ...... 75 Image Analyses...... 76 Statistical Analyses ...... 77 Acknowledgements ...... 77 Disclosure ...... 78 References ...... 78 Figures ...... 86

CHAPTER 3: HUMAN UMBILICAL CORD BLOOD CELLS PROTECT OLIGODENDROCYTES FROM BRAIN ISCHEMIA THROUGH AKT SIGNAL TRANSDUCTION ...... 99 Abstract ...... 100 Keywords ...... 100 Abbreviations ...... 101 Introduction ...... 102 Materials and Methods...... 103 Animal Care ...... 103 Mixed Glial Culture Preparation ...... 104 OL Culture Purification ...... 104 Oxygen Glucose Deprivation...... 105 LDH Assay ...... 106 RNA Collection and Purification ...... 107 Quantitative Real Time Polymerase Chain Reaction ...... 107 Laser Doppler Blood Flow Measurement ...... 108 Middle Cerebral Artery Occlusion ...... 109 HUCB Cell Treatment ...... 110 Immunohistochemistry ...... 110 Image Analyses...... 112 Statistical Analyses ...... 113 Results ...... 113 Akt Inhibition Negated HUCB Cell Protection In Vitro ...... 113 HUCB Cells Increased Akt Phosphorylation In Vitro ...... 114 HUCB Cells Induced Expression of Prdx4 is Suppressed by Akt Inhibition In Vitro ...... 114 Delayed HUCB Cell Treatment Increased Akt Phosphorylation in Cerebral White Matter after MCAO ...... 115 HUCB Cells Induce Prdx4 Expression in the External Capsule...... 115 P-Akt and Caspase 3 Co-localized with OL marker RIP ...... 116 HUCB Cell Treatment Reduces Caspase 3 Activation in the External Capsule Following MCAO ...... 116 Discussion ...... 117 Conclusions ...... 119 Acknowledgements ...... 120 Disclosure ...... 120 References ...... 121 Figures ...... 127

CHAPTER 4: LEUKEMIA INHIBITORY FACTOR AND GRANULOCYTE COLONY STIMULATING FACTOR INDUCE ANTIOXIDANTS TO ENHANCE SURVIVAL OF OLIGODENDROCYTES EXPOSED TO OXYGEN GLUCOSE DEPRIVATION ...... 134 Abstract ...... 135 Introduction ...... 135

iii

Materials and Methods...... 138 Animal Care ...... 138 Mixed Glial Culture Preparation ...... 139 OL Culture Purification ...... 139 Oxygen Glucose Deprivation...... 140 LDH Assay ...... 142 SOD Activity ...... 142 RNA Collection and Purification ...... 143 Quantitative Real Time Polymerase Chain Reaction ...... 143 Immunohistochemistry ...... 144 Image Analyses...... 146 Statistical Analyses ...... 147 Results ...... 147 Determination of Efficacious LIF Concentration ...... 147 Akt Inhibition Blocked LIF Protection ...... 147 OGD Increased Akt Phosphorylation ...... 148 LIF Increased OL Expression of Prdx4 and Mt3 in OL Cultures ...... 148 Akt Inhibitor IV Blocked LIF Induced Prdx4 Expression but Increased Mt3 Expression ...... 149 LIF Reduced SOD Activity in OL Cultures ...... 149 LIF Neutralizing Antibody Did Not Block HUCB Cell Effect ...... 150 GCSF Rescued OL from 24 hrs OGD ...... 150 Akt Inhibitor IV Blocked GCSF Mediated Effects...... 151 Discussion ...... 151 Acknowledgements ...... 156 Disclosure ...... 157 References ...... 157 Figures ...... 164

CHAPTER 5: DISCUSSION ...... 173 Overview ...... 174 HUCB Cells Rescued OLs ...... 176 HUCB Cells Enhanced Survival Protein Expression ...... 178 Mechanism by Which HUCB Cells Exert Beneficial Effects ...... 180 LIF Replaced HUCB Cell Treatment ...... 183 LIF Neutralizing Antibody Did Not Block HUCB Cell Effect ...... 185 GCSF Protected OLs Exposed to OGD ...... 186 Implications and Limitations ...... 187 References ...... 190 Figures ...... 199

ABOUT THE AUTHOR ...... End Page

LIST OF TABLES

Table 1: HUCB Cell Treatment Alters Gene Expression in OLs Subjected to 24 hrs OGD...... 97

Table 2: Common Transcription Factor Binding Sites Present in the Promoters of Upregluated Genes...... 98

LIST OF FIGURES

Chapter 2

Figure 1: OLs Differentiate Into the Mature Phenotype...... 86

Figure 2: HUCB Cells Decrease LDH Release From OLs Subjected to 24 hrs OGD...... 87

Figure 3: Affymetrix Gene Array Fold Changes are Confirmed by qRT- PCR ...... 88

Figure 4: HUCB Cells Reduce Infarct Volume ...... 89

Figure 5: HUCB Cells Rescue OLs of the External Capsule Following Ischemic Insult ...... 90

Figure 6: HUCB Cells Increase White Matter Uhmk1 Expression Following Ischemic Insult...... 91

Figure 7: HUCB Cells Alter White Matter Protein Expression Following Ischemic Insult...... 92

Figure 8: Immunohistochemical Quantification of White Matter Protein Expression ...... 93

Figure 9: Prdx4, Uhmk1, Insig1 and Mt3 Colocalized With OL Marker RIP ...... 94

Figure 10: Prdx4 is Expressed in Astrocytes but not Microglia/macrophages Following Ischemic Insult ...... 95

Figure 11: Mt3, Uhmk, and Insig1 are not Expressed in Microglia/macrophages or Astrocytes Following Ischemic Insult...... 96

Chapter 3

Figure 1: Akt Inhibition Negates the Protective Effects of HUCB Cells on Cultured OLs...... 127

Figure 2: HUCB Cells Increase Akt Phosphorylation in OLs Subjected to 24hrs OGD...... 128

Figure 3: Akt Inhibitor IV Suppresses HUCB Cell Induced Prdx4 Expression During OGD ...... 129

Figure 4: HUCB Cells Increase Akt Phosphorylation In Vivo ...... 130

Figure 5: HUCB Cells Induce Prdx4 Expression In Vivo ...... 131

Figure 6: Akt and Caspase 3 are Activated in OLs In Vivo ...... 132

Figure 7: HUCB Cells Reduce Caspase 3 Activation In Vivo ...... 133

Chapter 4

Figure 1: LIF Attenuate Stroke Induce OL Dysfunction ...... 164

Figure 2: Akt Inhibition Negates the Protective Effects of LIF on Cultured OLs ...... 165

Figure 3: OGD Increased Akt Phosphorylation in OLs Subjected to 24 hrs OGD...... 166

Figure 4: LIF Increased OL Expression of Prdx4 and Mt3 in OL Cultures .... 167

Figure 5: Akt Inhibitor IV Suppresses LIF Induced Prdx4 Expression but Increased Mt3 expression ...... 168

Figure 6: LIF Reduced SOD Activity in Primary OL Cultures ...... 169

Figure 7: LIF Neutralizing Antibody Did not Block HUCB Cell Effect ...... 170

Figure 8: GCSF Rescued OLs from 24 hrs OGD ...... 171

Figure 9: Akt Inhibitor IV Blocked GCSF Mediated Effects ...... 172

vii

Chapter 5

Figure 1: Mechanisms of OL Cell Death Following Ischemia and the Effect of HUCB cells ...... 199

Figure 2: Mechanisms of HUCB Cell Protection of OLS ...... 200

Figure 3: Mechanisms of LIF Protection of OLs Subjected to Ischemia ...... 201

viii

ABSTRACT

Oligodendrocytes (OL)s are the dominant cell type in the white matter and are integral for synaptic transmission essential for proper neuronal communication between brain areas. Previous studies have shown that intravenous administration of the mononuclear fraction of human umbilical cord blood (HUCB) cells in rat models of stroke reduced white matter injury, gray matter injury and behavioral deficits. Yet the mechanisms used by HUCB cells remain unknown in ischemic injury. These studies will investigate both in vitro and in vivo approaches to elucidate this mechanism in OLs. When mature primary OLs were coincubated with HUCB cells, HUCB cells secreted soluble factors that reduced cell death in OLs exposed to OGD. Microarray analysis revealed that HUCB cell treatment induced OL gene changes. These changes included genes involved in cell proliferation, signaling, anti-oxidant activity, and myelination. To extend these findings, the middle cerebral artery occlusion

(MCAO) model was used to assess the expression profile of protein products of gene changes observed in vitro. The in vivo data mirrored in vitro data in that metallothionein 3 (Mt3), peroxiredoxin 4 (Prdx4), myelin oligodendrocyte glycoprotein (Mog), U2AF homology kinase 1(Uhmk1), and insulin induce gene

1(Insig1) were upregulated in OLs of the white matter tract adjacent to the infarct.

Furthermore, double immunofluorescence staining determined that OLs

expressed these proteins. Other reports have shown that HUCB cells secrete soluble factors related to cellular protection, including interleukin 6 (IL-6), interleukin 8 (IL-8), and interleukin 10 (IL-10). Other factors are known for their proliferative actions, such as vascular endothelial growth factor (VEGF), BDNF, platelet derived growth factor B (PDGF-B), leukemia inhibitory factor (LIF), and granulocyte colony stimulating factor (GCSF) all of which converge on the Akt survival pathway. Given these findings we hypothesize that Akt activation is integral to HUCB cell mediated OL protection. In models of excitotoxicity, the addition of Akt inhibitor IV blocked HUCB cell mediated protection in OL cultures exposed to 24 hrs OGD. In vivo, HUCB cell treatment increased Akt activation, antioxidant protein expression and decreased caspase 3 cleavage in the external capsule in a time dependent manner. The next series of experiments determine whether the soluble factors secreted by HUCB cells could replace HUCB cells as treatment. LIF expression is increased in HUCB cells as compared to peripheral blood and as previously mentioned, LIF is secreted by HUCB cells. Additionally,

LIF rescued OLs from spinal cord and experimental autoimmune encephalomyelitis injury. Thus LIF was investigated. LIF protected OL subjected to 24 hr OGD, increased antioxidant Prdx4 gene expression and reduced reactive oxygen species production. Additionally the inclusion of Akt inhibitor IV blocked LIF induced OL protection. Similar results were obtained when GCSF was evaluated. All these findings indicate that HUCB cell mediated OL/white matter protection is due to the soluble factors secreted by the mononuclear population of these cells. These soluble factors including LIF activate cellular

x machinery leading to enhanced cellular survival. Here we found a specific survival pathway activated by soluble factors released from HUCB cells, leading to Akt activation. Akt activation arrests stroke induced apoptosis and reduced the expansion of the infarct, promoting functional recovery from acute ischemic injury.

CHAPTER ONE

INTRODUCTION

D.D. Rowe, MS, A.E. Willing, PhD, K.R. Pennypacker PhD.

Overview of Stroke

Each year an estimated 780,000 Americans will experience an initial ischemic event, and over 150,000 of these individuals will succumb to complications as a direct result of the stroke (Association 2008). According to the

American Heart Association, stroke is the third leading cause of death in the

United States. The high incidence of stroke places an enormous financial burden on the American health care system, an estimated $69 billion yearly (Lloyd-

Jones, Adams et al. 2009). Therefore the development of effective therapeutics to reduce stroke related morbidity and mortality will provide both financial and societal benefits.

Tissue plasminogen activator (t-PA) is currently the only Food and Drug

Administration (FDA) approved treatment for acute ischemia (Pan, Yu et al.

2008). t-PA represents a class of thrombolytic agents which restores blood flow to ischemic areas by enzymatically dissolving clots, but does not possess neuroprotective activity. In clinical trials, t-PA treatment within 4 hours of stroke onset significantly reduced stroke-related morbidities; use beyond this limited therapeutic time window is ineffective and could prove detrimental (Hacke,

Donnan et al. 2004). As a result, only 3-5% of stroke patients are able to use t-

PA (Marler and Goldstein 2003; Association 2008). Effective stroke therapies are clearly needed to extend the therapeutic time window providing treatment for a larger population of stroke patients.

Types of Stroke

Ischemic Stroke

The major classifications of stroke are defined as either hemorrhagic or ischemic. Ischemic strokes account for 83% of all strokes and occur when an obstruction is present within a vessel supplying blood to the brain (Association

2008; Lloyd-Jones, Adams et al. 2009). Depending on origin and location of the clot, an ischemic event can be further classified as thrombotic or embolic. With thrombosis, ischemia develops at the point of the clot, whereas in an embolic stroke the clot arises from a specific location, travels in the circulatory system and is lodged in a narrowed vessel (Association 2008). The severity of a stroke is further based on cerebral blood flow (CBF) downstream the obstructed vessel.

The terms acute, mild, or severe are used to describe the extent of an ischemic injury. Normal CBF is typically recorded as 45-110 cm3 × 100 g-1 × min-1. An

acute ischemic stroke is marked by immediate depletion of oxygen and glucose

in the affected brain tissue, while mild ischemia is classified as a condition in

which a residual CBF of 11–20 cm3 × 100 g-1 × min-1 is recorded. This reduction

in CBF leads to the activation of reversible apoptotic pathways, the target of

developing therapeutic agents. In severe ischemic events, CBF of ≤ 6 cm3 × 100

g-1 × min-1 is usually recorded; this condition is classically associated with

complete energy loss in affected cells, leading to necrosis (Kaufmann, Firlik et al.

1999). Given that immediate cell death as a result of severe ischemia is not

reversible, neuroprotective agents will not reduce these infarctions, but may

target behavior deficits that result from the ischemic insult.

Hemorrhagic Stroke

Accounting for 17% of all strokes, hemorrhagic strokes result from ruptured blood vessels leading to severe bleeding, swelling and compression of tissue in the affected area. Hemorrhagic strokes are subcategorized depending on the location of the hemorrhage; intracerebral and subarachnoid hemorrhages.

Intracerebral hemorrhages are the result of ruptured vessels leading to bleeding within these brain areas, the thalamus, basal ganglia, cerebellum or the pons.

Subarachnoid hemorrhages denote bleeding from a ruptured vessel into the space between the brain and the skull (subarachnoid space). Hemorrhagic strokes usually result from weakened blood vessels as a consequence of aneurysms, head injuries, high blood pressure and arteriovenous malformations

(Association 2008). Because of the pervasive and devastative nature of ischemic injury in the current population, this topic will be extensively discussed.

Ischemic Stroke Pathology

Energy Depletion

The brain rapidly metabolizes oxygen and glucose. As a result, immediate energy depletion occurs following the occlusion of vessels supplying blood to the brain. Within minutes of energy starvation, cellular ATP synthesis is arrested.

Lack of energy production leads to an imbalanced cellular ion gradient and cellular excitotoxicity due to membrane depolarization, sodium influx, calcium influx, and potassium efflux into the extracellular space (Doyle, Simon et al.

2008). These processes trigger the degradation of membranes and proteins

essential to the maintenance of homeostasis resulting in the outward radiation of

cellular depolarization from the ischemic core leading to calcium accumulation

within cells. Elevated intracellular calcium and sodium increase mitochondrial

reactive oxygen species (ROS) production, cellular reactive nitrogen species

(RNS), free radical production and activation of cell death cascades (Coyle and

Puttfarcken 1993; Doyle, Simon et al. 2008).

Immune Response

Negligible amounts of proinflammatory cytokines are present in the brain under normal physiological conditions. Energy depletion as a result of ischemia triggers activation of the immune system. Once activated, the resident central nervous system (CNS) immune cells, the microglia, secrete cytokines,

chemokines, matrix metalloproteinases (MMP), nitric oxide (NO) and reactive

oxygen species (ROS) creating an environment conducive to the recruitment of

proinflammatory cells (Sharma and Kumar 1998; Wang, Tang et al. 2007).

Recruitment of these cells increases the secretion of proinflammatory mediators

resulting in the disruption of the blood brain barrier, infiltration of peripheral

inflammatory and immune cells such as leukocytes. Microglia along with

peripheral immune cells accumulate in the infarct area where the inflammatory

signal is amplified, exacerbate injury and perpetuate the destructive cycle leading

to the outward expansion of the infarct (Emsley and Tyrrell 2002; Danton and

Dietrich 2003).

Reperfusion

Stroke pathology is not only a consequence of ischemia which causes oxygen and glucose deprivation, but also a result of reperfusion. The reperfusion

of an occluded vessel increases cellular ROS. ROS are produced by

dysfunctional mitochondria that generate oxidative stress and release apoptosis related proteins upon tissue oxygenation. ROS initiate proinflammatory cytokine

and chemokine production by immune cells. In response to proinflammatory

cytokines, inflammatory cells release MMPs, NO and ROS. This reperfusion led

inflammatory response results in blood brain barrier disruption, infiltration of

peripheral immune cells and expansion of the infarct (Emsley and Tyrrell 2002;

Danton and Dietrich 2003).

As pointed out, both ischemic insult and reperfusion results in delayed cell

death by disrupting the blood brain barrier allowing infiltration of peripheral

immune cells. The initial mechanisms of both injuries are different but converge

on the modulation of inflammation to amplify and exacerbate disease pathology.

Therefore animal models used for the development of stroke therapeutics should

aim to eliminate the over production of these proinflammatory mediators.

Animal Models of Stroke

An assortment of animal models to evaluate stroke related pathology has

been developed to assess new therapeutics. Importantly, basic research aims

specifically to translate beneficial effects of developing therapeutics from the

laboratory to clinical trials involving humans. Three basic animal models are

currently utilized in stroke research: global ischemia, focal ischemia and hemorrhagic injury (Richard Green, Odergren et al. 2003). In hemorrhagic animal

models, bleeding is induced in a specific brain region depending on the

hemorrhagic classification (intracerebral or subarachnoid). Global and focal

ischemias are the most commonly used animal models; therefore these topics

will be discussed further.

Global Ischemia

Global ischemia involves the blockage of one or more major arteries

resulting in significant CBF reduction in areas perfused by the occluded vessel,

thus mimicking ischemic injury (Traystman 2003). Global ischemia can be

performed by varying techniques including bilateral carotid occlusion, two vessel

occlusion plus hypotension, or a four vessel occlusion (Richard Green, Odergren

et al. 2003). Bilateral carotid occlusions are achieved using an incision to expose

both carotid arteries, the vessels are isolated and ligated most commonly using a

silk suture (Fujishima, Morotomi et al. 1979).

In the permanent occlusion model, the occlusion/ligation is permanent; in

the transient model, the method of occlusion is reversed following an allotted

time to restore blood flow. Use of the permanent model aims to investigate stroke

induced infarct, while the transient model is used to investigate both stroke

induced infarct and reperfusion injury.

Two vessel occlusion plus hypotension employs the same technique as

described in bilateral carotid occlusion, but hypertensive animals are used. This

7 model intends to examine the cumulating effects of both high blood pressure and ischemia. Four vessel models incorporate the bilateral carotid model, but also added the occlusion of two vertebral arteries (Pulsinelli and Brierley 1979). This procedure calls for the occlusion of all four major arteries that supply blood to the brain. This method produces a significant decrease in CBF resulting in global ischemia (Pulsinelli and Brierley 1979). Although the four vessel occlusion model restricts all major vessels supplying the CNS, blood still reaches the brain through the collateral and anterior spinal arteries in the para vertebral and cervical muscles (Pulsinelli and Buchan 1988). Therefore a complete global ischemia is not achieved in the models mentioned, although the four-vessel model produces the most significant reduction in CBF. Myocardial infarctions are the only method used to produce a complete global ischemia. Although effective and easily replicated, global ischemia is not comparable to the human model of stroke. Global ischemia is less relevant to the human stroke condition because global ischemia is not a common feature of human stroke, but does have a place in the study of cardiovascular incidents. In the human population, focal ischemia is the major injury pattern observed (Traystman 2003).

Focal Ischemia

Focal ischemia entails the obstruction of a specific vessel, creating an infarct in areas perfused by the vessel. This model is primarily achieved by obstructing the middle cerebral artery; a procedure which reduces CBF mainly to the striatum and cortex (Garcia, Liu et al. 1995; Kanemitsu, Nakagomi et al.

2002). Middle cerebral artery occlusion (MCAO) is achieved by isolating both the

external and the common carotid, from which the MCA is obstructed either by the

introduction of a filament, suture or photothrombosis (Carmichael 2005). In the

filament model, a line is introduced at the base of the internal carotid artery and advanced into the MCA. In the suture model, a silk thread is used to tie off the internal carotid artery, thus restricting blood flow to areas perfused by the MCA.

On the other hand, the photothrombosis model involves the use of photooxidation to develop a clot in the target vessel, providing the necessary occlusion resulting in reduced CBF.

Following MCAO, cell death is rapid in the striatum and cortex, and the expansion of the infarct via apoptotic cascades are time dependent (Garcia, Liu et al. 1995; Carmichael 2005). Infarction areas are thus relative to MCAO duration. MCAO induced striatum and cerebral cortex dysfunction produces motor, autonomic and cognitive deficits (Carmichael 2005). These deficits are a result of stroke led degeneration of areas integral to the execution of such functions. In humans, occlusion of the middle cerebral artery is the most common cause of an ischemic event, therefore occlusion of the MCA in animal models serves to develop specific treatments that would benefit a large population of patients (Traystman 2003). Like models of global ischemia, the MCAO model of focal ischemia can be transient or permanent. In the transient model, the vessel is obstructed for a period of time, followed by removal of the obstructive force, where in the permanent model the obstruction is not removed. Ischemia plus reperfusion is studied in the transient model, where only the ischemic damage is

9 accessed in a permanent occlusion model (Richard Green, Odergren et al.

2003).

All models mentioned produce global or focal ischemia. These models provide reproducible effects tailored towards the research of specific investigators. Each model provides benefits and drawbacks and is used specifically for the length of time required for the ischemic damage as it correlates to the region of the brain affected.

Moving Past the Neuron-Centric Perspective

A primarily neuron-centric approach has influenced the past decade of stroke related research. Specifically, this movement is primarily aimed at neuronal preservation and stabilization following ischemic injury. This neuron- centric approach has led to an overwhelming proportion of research focused at limiting ischemic damage to the gray matter; thus the current literature is saturated with experimentations studying the effects of stroke on gray matter. As a result, very little has been published investigating white matter pathology although it plays a significant role in the debilitating disease that is stroke. The white matter, like the gray matter, is significantly impacted by ischemic brain injury. Cerebral white matter accounts for 50% total brain volume in humans, oligodendrocyte (OL) are the major cell type of the white matter and are integral in proper brain function (Miller, Alston et al. 1980). The significance of this region has contributed to an emerging consensus highlighting the importance of OLs

and the white matter in research aimed at limiting functional deficits resulting

from stroke induced pathology (Arai and Lo 2009).

White Matter

Overview

By volume, the white matter account for 50% total brain volume in humans

and is responsible for efficient synaptic transmission and proper cerebral function. It is of no surprise that white matter damage can be more devastating than comparable gray matter injury when similar size areas are affected by

stroke (Goldberg and Ransom 2003). Mature OLs of the cerebral white matter

myelinate the central nervous system (CNS). The myelin produced by OL,

ensheaths neuronal axons to facilitate fast and efficient synaptic transmission.

OL Development

OL progenitors arise in vivo from cells of the subventricular zone of the

lateral ventricles, differentiate, then migrate to regions of developing white and

grey matter until reaching the appropriate axons (Levison and Goldman 1993).

After reaching their target axons OLs exit the cell cycle, become non-migratory

and differentiate into myelin producing OLs. During maturation, OLs begin to

down regulate precursor OL proteins including platelet derived growth factor

alpha receptor (PDGF-AA) and NG2 chondroitin sulfate proteoglycan and induce

the expression of proteins, such as myelin basic protein (MBP) (McTigue and

Tripathi 2008). Upon beginning the expression of myelin proteins, OLs have fully

developed into the mature phenotype.

Myelination occurs in the following four stages: the recognition and

adhesion of an OL to an axon, synthesis and transport of myelin to axon,

wrapping of myelin around axon, and finally compaction of myelin sheath. During

the myelination process, only axons with a diameter greater than 0.2µm are

ensheathed. Unlike Schwann cells of the peripheral nervous system that

myelinate axons at a 1:1 ratio, OLs have the ability to myelinate 40-60 different

axons simultaneously. During the active phase of myelination, each OL

produces an estimated 5.50 X 103 µm2 of myelin membrane surface area per day, a process requiring a large expenditure of energy (Pfeiffer, Warrington et al.

1993; Taveggia, Zanazzi et al. 2005; Simons and Trajkovic 2006).

Myelin sheaths are formed by the coiled wrapping of glial plasma membrane extensions around axons followed by the extrusion of cytoplasm and the compaction of the stack membrane bilayers. Myelin sheaths are rich in glycosphingolipids and cholesterol to provide insulation of axons to maximize

conduction velocity (Simons and Trajkovic 2006). By weight, myelin is composed

of 30% protein and 70% lipids; the two major protein classes are MBP and

proteolipid proteins (PLP). Minor proteins such as CNPases, myelin associated

glycoprotein (MAG), and myelin oligodendrocyte glycoprotein (MOG) are also

present (Morell, Greenfield et al. 1972; Larocca and Rodriguez-Gabin 2002). In addition, proteins are synthesized in different regions based on function. MBP and myelin oligodendrocyte associated basic proteins (MOABP) are synthesized

12 close to the site of myelin assembly (Colman, Kreibich et al. 1982; Barbarese,

Koppel et al. 1995; Gould, Freund et al. 1999). PLP and MAG are synthesized in the endoplasmic reticulum, then transported by vesicles to the Golgi. From the

Golgi these proteins are transported via COPI and COPII to the forming myelin sheath. Cooperatively, both the minor and major proteins of myelin function together to maintain the myelin sheath, and the absence of one could be destructive to the sheath stability (Townsend and Benjamins 1983; Bizzozero,

Soto et al. 1984).

In addition to myelination, OLs serve an important role supporting the survival and function of neighboring neurons. OLs regulate axonal size, ion channel clustering such as sodium (Na+) and potassium (K+) and also produce trophic factors including insulin like growth factor 1 (IGF-I), nerve growth factor

(NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5) (Du and Dreyfus 2002). Additionally, failures and abnormalities in myelination are implicated in disruptions of normal impulse conduction and interference with neurological function and can potentially lead to neuronal loss (Noble 2005 ). The importance of myelination is evident not only in demyelination diseases such as multiple sclerosis, but also in mice lacking the major proteolipids of myelin. These animals tend to experience axonal swelling and degeneration (Du and Dreyfus 2002).

Stroke and Oligodendrocytes

CNS myelination is the primary function of white matter OLs. As a result, the primary consequence of OL death is axonal demyelination. Axonal demyelination leads to delays or complete blockage of axonal conduction, which results in impaired neuronal signaling and cell death. OLs are also responsible for normal axonal transport as well as microtubule levels. Lack of myelin contributes to elevated microtubule numbers but a sharp decrease in microtubule stability (McTigue and Tripathi 2008). Additionally, OLs like neurons and astrocytes are known to have very high metabolic rates. These high metabolic demands render OLs inherently susceptible to ischemia. Naturally, of the different types of glia, OLs were found to be the most prone to hypoxic and/or glucose deprived conditions (Lyons and Kettenmann 1998). OL injury is important as it relates to the treatment of stroke. Therefore it is important to understand stroke induced OL dysfunction that treatments can be tailored to maintenance of cerebral white matter following injury.

Oxidative Stress

Oxidative stress is one of the most frequent causes of stroke induced OL dysfunction. Cellular stress is increased during anaerobic respiration after ischemia, particularly during reperfusion (Dewar, Underhill et al. 2003). Of the compounds produced in OGD environments, ROS and RNS are the most detrimental to cellular homeostasis. ROS are natural by products of oxygen metabolism and are metabolized into benign compounds under normal

physiological condition. Yet, under biological stress, ROS are generated to toxic levels causing cellular dysfunction (Allen and Bayraktutan 2009). The brain is especially prone to ROS induced damage, the abundance of peroxidizable lipids and natively high oxygen consumption are the main culprit. In the in vitro stroke model OGD, aerobic respiration is disrupted resulting in the accumulation of respiratory chain intermediates allowing electron leak to generate ROS

(Abramov, Scorziello et al. 2007). Superoxide is the primary ROS from which other more reactive ROS are generated. Superoxide generates hydrogen peroxides, which generates hydroxyl radicals. RNS are also generated from the interaction of nitrogen and superoxide. These intermediates include nitric oxide, peroxynitrous acid that produces hydroxyl radical. The accumulation of these radicals depends on the oxidizable substrate, availability of oxygen and antioxidant enzymes (Allen and Bayraktutan 2009).

OLs are susceptible to oxidative stress due to the large amount of energy necessary to synthesize and maintain myelin. Myelination is an energy dependent process and requires large amounts of ATP and oxygen consumption.

As described above, OGD leads to overproduction of superoxide leading to hydrogen peroxide and ROS byproducts which if not metabolized cause OL deoxyribonucleic acid (DNA) damage, lipid per-oxidation and apoptosis. OL susceptibility to oxidative stress is also a result of myelin synthetic enzymes that requires co-factors such as iron. Of cerebral cell types, OLs possess the largest intracellular iron stores. Iron is highly reactive, when oxidized it reacts vigorously resulting in to free radical formation (McTigue and Tripathi 2008). For example,

hydroxyl radical is generated from hydrogen peroxide by ferrous iron that has

been reduced by superoxide (Allen and Bayraktutan 2009). Following ischemic injury, free radical formation as a result of iron oxidation is rapidly increased causing lipid peroxidation. In conjunction with elevated iron levels, OLs possess

low anti-oxidants stores. Glutathione is one such anti-oxidant (McTigue and

Tripathi 2008). Relative levels of antioxidants are important to maintain the

balance of ROS without accumulating enough for a toxic effect. OLs low anti-

oxidant stores encourage rapid accumulation of ROS leading to accelerated

cellular dysfunction.

Additionally, sphingolipids are a major component of myelin synthesis and

play a damaging role in OL injury. Sphingolipids contain ceramide cores that

coalesce to form domains enriched in death receptors following injury. These

death receptors include CD95, tumor necrosis factor receptor. In addition, the

clustering of ceramide cores increases the sensitivity of OLs to death receptor

signaling. But this represents only one way in which ceramides contribute to OL

injury. Ceramides can also be released intracellularly by enzymatic cleavage of

membrane sphingolipids by sphingomyelinase (SMase). This enzyme is

normally inactive but is activated during CNS injury such as stroke (McTigue and

Tripathi 2008).

Excitotoxicity, in which α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid (AMPA)/Kainate receptors are over stimulated, also plays a role in ischemic

OL death. These receptors are stimulated as a result of glutamate, excessive

stimulation of these receptors leads to elevated intracellular calcium. Elevated

16 intracellular calcium initiates cascades activating enzymes that degrade cytoskeleton proteins causing mitochondrial disruption leading to ROS production and cytochrome c release (McDonald, Althomsons et al. 1998). Following an ischemic event, as stated above, a variety of cytokines and chemokines are released in the cellular space by a variety of cells. The cytokines interleukin 1 beta (IL-1β), interleukin 2 (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF-α) have all been shown to promote OL death directly or indirectly (McTigue and Tripathi 2008). TNF-α and IFNγ signaling induce free radical production, which results in OLs cell death through oxidative stress as previously described. TNF-α can also directly kill OLs by binding to the p55 TNF receptor which initiates DNA degradation and caspase independent apoptosis

(McTigue and Tripathi 2008).

With the mechanisms of white matter injury outlined above, it is important to tailor treatment specific to arrest these processes. Few in vitro treatments are efficacious in vivo, and a minute number of these successful treatments are assessed in clinical trials. Below, treatments that have attained some promise in clinical trials will be discussed.

Clinical Trials

Treatment following an ischemic event does not aim to affect the immediate necrosis at the core of the infarct, but to prevent the expansion of the infarct core. Chemicals identified as potential stroke treatment are first examined in a laboratory setting beginning with in vitro stroke models. OGD is the most

commonly used in vitro stroke model, where cellular media is devoid of glucose

and cells are exposed to an oxygen deficient environment for an extended period

of time. If in vitro trials are successful, in vivo studies are performed using animal models, after which, viable therapeutics are scheduled for human clinical trials following FDA approval. Over 150 clinical trials investigating therapeutic agents targeting ischemic injury have been performed. These therapeutics are classified as nonpharmacologic, antithrombotic and neuroprotective agents, all but one resulted in failure (Kidwell, Liebeskind et al. 2001). A successful drug is defined as a drug approved by the FDA for treatment. So far the only such existing drug is the thrombolytic agent t-PA. With limited patients qualifying for treatment with t-

PA, basic scientists like clinicians have recognized the urgent need for new

therapeutics to combat the ever-growing disease that is stroke. As a result over recent decades, not only have clinical trials increased, but the sample size attributed to each trial has also increased (Kidwell, Liebeskind et al. 2001).

Neuroprotective Agents

Neuroprotective trials examined drugs that promote membrane repair, free radical scavengers, antiedema, metabolic activations, modulators of calcium influx, and modulators of excitatory amino acids (Kidwell, Liebeskind et al. 2001).

Many of these drugs were successful in vitro and showed promise in several in vivo animal models. Yet, the cellular protection and behavioral recovery observed in vivo has not transferred to human clinical trials. The lack of success in human trials could be attributed to the controlled environment in which animal studies

18 are performed. In animals, specific stroke models elicit damage in specific areas of the brain. Additionally, the success of a drug is usually based on the action of treatment in specific brain areas; therefore an overall protective view including behavioral recovery is often overlooked. Also the side effects of these drugs are usually not completely understood or evaluated (Richard Green, Odergren et al.

2003). An example of such a neuroprotective agent is the use of nimodipine, a calcium antagonist. Nimodipine specifically blocks the influx of calcium following ischemia, thus arresting excessive cellular stimulation and cell death attributed to excitotoxicity (Steen, Gisvold et al. 1985). Nimodipine was successful in vitro and in animal models at reducing cell death and infarct volume respectively, but outcomes following ischemic injury were not improved in human trials (Steen,

Gisvold et al. 1985; Horn, de Haan et al. 2001). In addition, nimodipine had an unintended side effect of lowering patient’s blood pressure and have been shown to worsen outcome following stroke. This side effect was identified only after the drug was evaluated in human trials (Ahmed, Nasman et al. 2000; Horn, de Haan et al. 2001; Richard Green, Odergren et al. 2003).

Addressing Failure

Nimodipine, like other neuroprotective and nonpharmacologic drugs, experienced success in animal studies but failure in human clinical trials. The high failure rate of treatment for ischemic injury could be attributed to the monotherapy performed in clinical trials and the neuron-centric view. Stroke is a multicellular disease meaning the entire CNS is affected following an ischemic

insult. Neurons and glial cells are adversely affected by ischemia, and responds

in varying ways. Thus, diverse cellular populations are affected in an ischemic

attack. Therefore different pathways and substrates contribute to the expansion

of the infarct and the final outcome of the disease. A narrow focus on the gray

matter to combat stroke pathology is naive. A broad view addressing both gray

and white matter injury as a result of stroke is necessary to the development of

new therapies.

Antithrombotics

Antithrombotic trials evaluate agents that increase blood flow, many of

which are anticoagulants, fibrinolytic and antiplatelet compounds (Kidwell,

Liebeskind et al. 2001). This area of study, thus far, has been the most

successful, with the advent of t-PA, the only FDA approved treatment for ischemic injury.

Tissue Plasminogen Activator

t-PA was approved by the U.S Food and Drug Administration (FDA) in

1996, and is still the only thrombolytic drug approved as treatment for an acute

ischemic injury (Pan, Yu et al. 2008). t-PA is a serine protease enzyme that

converts plasminogen to plasmin. Thrombotic clots are formed from fibrin and

platelets by the body to inhibit blood loss, when these clots enter the circulatory

system a thrombosis can result. t-PA works by cleaving plasminogen into

plasmin, this cleavage activates plasmin which cleaves fibrin (Slaets, Dumont et

20 al. 2008). Thus, t-PA alike other thrombolytics works by dissolving clots, and therefore restores blood flow to ischemic areas. t-PA treatment within 3 hrs of stroke onset significantly reduces the effects of stroke (Hacke, Donnan et al.

2004). This cited study also reported that benefits were also seen 4 hrs following stroke but negligible to no benefit was observed when given 6 hrs post stroke.

Although beneficial, usage of this treatment increased the patient’s risk of a possible hemorrhagic event (Wardlaw, Warlow et al. 1997). The American Heart

Association reports that only 3-5% of stroke patients employ this treatment, based on the time qualifications and the specificity of treatment only to ischemic injury. To qualify for t-PA treatment, a patient must first recognize the onset of the stroke, get to the hospital, have a CT scan performed to specify that the stroke is ischemic in nature and then receive treatment if all required criterions are met.

The most stringent of these qualifications is the 3 hrs time window within which a patient must be evaluated and deemed eligible for treatment (Marler and

Goldstein 2003).

Stem Cells

Unlike previously discussed therapeutics, stem cell therapy first aimed to reduce the progression of stroke pathology by replacing neurons that have succumbed to the ischemic insult, and promote neurogenesis while extending the therapeutic time window for treating strokes. Unlike other cell replacement therapies, stem cell therapies initially aimed to generate and replace not just one specific cell type, but a large and varying population of cells. Thus far various

sources of stem cells have been tested to improve function following transplantation in animals subjected to stroke. Promising results were shown using a mouse neuroepithelial stem cell line, human NTera-3 cell line and human

bone marrow/mesenchymal stem cells (Fujishima, Morotomi et al. 1979).

Ischemia potentiates the secretion of neurotrophins and bioactive factors by

mesenchymal stem cells (MSC) (Oh, Fujio et al. 1998). In animal studies, MSCs

released bioactive factors in the ischemic boundary, reducing cellular apoptosis

and induced cellular proliferation. Regeneration was observed, where newly formed cells expressed proteins identifiable to progenitor like neuron and astrocytes (Chen, Li et al. 2003). Although cellular recovery has been identified in

vivo following stem cell treatment, evidence shows very little of the transplanted

cells survive and migrate to the brain (Fujishima, Morotomi et al. 1979). Thus, it

is now evident that stem cells do not replace dysfunctional cells in the ischemic

boundary, but promote repair and neurogenesis.

Stem cell transplantation in human trials has shown some success; this

included the use of human neuronal cells derived from a human teratocarcinoma

line and fetal porcine cells. The use of human teratocarcinoma showed

improvements in patients tested on the European stroke scale, but in phase II

trials, no improvement in outcome as tested by the European Scale after six

months was reported although improvements were observed in the Action Arm

Research Test and the Everyday Memory Test (Kondziolka, Wechsler et al.

2000; Bang, Lee et al. 2005). A small study using fetal porcine cells was quickly

terminated by the FDA after patients reported seizures and increased

neurological deficits weeks following transplantation (Marler and Goldstein 2003).

The most promising of the stem cell study involved the use of autologous marrow

stem cells (aMSCs). In experiments where aMSCs were administered

intravenously 4-5 or 7-9 weeks following initial onset of stroke in humans,

behavioral recovery was reported using two different exams. When compared against control, aMSC treated patients did not show a reduction in infarct volume but improvements were observed in both the Barthel index and modified Rankin scale (Bang, Lee et al. 2005). Both Barthel and modified Rankin scales are used to measure performance in basic activities of daily living, assessing the likelihood of being able to live at home independently following discharge from the hospice

(Mahoney and Barthel 1965; van Swieten, Koudstaal et al. 1988). Distinctly, the

modified Rankin scale is the most widely used clinical outcome measure for

stroke.

Human Umbilical Cord Blood Cell

Origin

Human umbilical cord blood (HUCB) cells represent a cellular population

taken from the placental umbilical cord of newborns. HUCB cells are easily

retrieved and available unlike cells such as bone marrow that requires the use of painfully extraction techniques to access the cells. Furthermore, because of the characteristic immaturity of these cells, there is a lower incidence of graft versus host disease following cell transplantation compared to other cell based therapies

(Lewis 2002). An additional benefit is the ability of HUCB cells to be

cryopreserved for later use, HUCB cells are effective three years following initial cryopreservation (Goodwin, Grunzinger et al. 2003). The mononuclear fractions of HUCB cells are typically used in therapy. This mononuclear fraction is composed of 77% lymphocytes, 23% monocytes and less than 1% CD34 hematopoietic stem cells. The lymphocyte population contains approximately

47% T lymphocytes. (Pranke, Failace et al. 2001).

Cellular Protection

The use of HUCB cells as a therapeutic treatment for stroke is a growing field of study, especially with the initial success of laboratory trials. OLs co- incubated with HUCB cells and exposed to OGD showed reduced activated caspase 3 immunoreactivity compared to untreated cells. In vivo, white matter

bundles were preserved in animals treated with HUCB cells compared to MCAO

groups not treated with HUCB cells (Hall, Guyer et al. 2009). Moreover, following

MCAO, HUCB cells migrate to the site of injury to reduce infarct volume and

improve behavioral recovery (Vendrame, Cassady et al. 2004; Newman, Willing

et al. 2005; Hall, Guyer et al. 2009). The cytokine monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein (MIP-1α) are responsible for these chemotactic effects promoting HUCB cell migration to the ischemic area ipsilateral to the infarct (Jiang, Newman et al. 2008). In the assessment of behavioral recovery, intravenous administration of HUCB cells 48 hrs post MCAO significantly improved motor recovery as measured by the step

test and accelerated rotorod test one-month post stroke (Newcomb, Ajmo et al.

2006).

Anti-inflammatory Effects

HUCB cells not only exert effects on the brain of an ischemic animal, but

also modulate the immune system. Administration of HUCB cells reduced stroke

induce pro inflammatory response by blocking the infiltration of peripheral

immune cells such as leukocytes (Vendrame, Gemma et al. 2005). This

reduction of microglia, macrophage and B cells recruitment to the infarct reduced

the production of pro inflammatory mediators TNF-α, IL-1β and IL2 expression

following stroke (Vendrame, Gemma et al. 2005). HUCB cell modulation of pro

inflammatory effectors was also evident in the spleen. Post MCAO, About 1% of

HUCB cells were found to migrate to the spleen following intravenous treatment

(Chen, Sanberg et al. 2001). While, splenectomy before MCAO reduced the activation of both peripheral and resident CNS immune cells in the brain. The reduction of immune cell activation arrested stroke pathology, where reduction in flouro jade positive cells was evident in the infarct area (Ajmo, Vernon et al.

2008). These experiments identified pleio-effects of HUCB cells; HUCB cells act

at the point of the CNS but also modulate and suppress the immune response

following an ischemic insult.

HUCB Cells Secrete Soluble Factors

The reported protective effects following HUCB cell systemic injection post ischemia is attributed to the soluble factors secreted by these cells. Upon stimulation of HUCB cells by epidermal growth factor/ fibroblast growth factor-2

(EGF)/ (FGF-2) or nerve growth factor/retinoic acid (NGF/RA), a vast number of soluble factors were secreted by HUCB cells. These factors included interleukins, growth factors, chemokines and metalloproteinase inhibitors. Many of the secreted factors are known for their pro-survival effects, including interleukin 6

(IL-6), interleukin 8 (IL-8), and interleukin 10 (IL-10) (Neuhoff, Moers et al. 2007).

Other factors are known for proliferative effects, such as vascular endothelial growth factor (VEGF), BDNF, platelet derived growth factor B (PDGF-B), leukemia inhibitory factor (LIF), and granulocyte colony stimulating factor (GCSF) among others and are known to activate survival pathways including phosphoinositide 3 kinase (PI3)/Akt (Kulik, Klippel et al. 1997; Jin, Mao et al.

2000; Newman, Willing et al. 2006; Neuhoff, Moers et al. 2007). The aforementioned findings established HUCB cell treatment as a possible therapeutic candidate for delayed treatment of ischemic injury. Yet, there are strong concerns with regards to the use of HUCB cells; these concerns include the ethical use of cell based therapy. Therefore, it is of utmost importance to investigate and find soluble factors that emulate the effects of HUCB cells; LIF represents one such soluble factor.

Leukemia Inhibitory Factor

Here we will discuss LIF, a pleiotropic cytokine secreted by HUCB cells.

LIF is a 180 amino acid single 4-α-helix glycoprotein (Kurek 2000; Metcalf 2003) that works through the LIF cell surface receptor complex which includes the LIF receptor β (LIFR) and the gp130 receptor chain (Kurek 2000). Cells throughout the body express LIFR. While LIF specifically interacts with the LIFR, it binds at a low affinity, but this affinity is increased once LIF is bound to the gp130 (Gearing,

VandenBos et al. 1992; Kurek 2000). Following LIF binding, LIFR and gp130 dimerizes and activates the gp130 intracellular signaling cascade. The activation of gp130 results in the activation of the Janus kinase signal transducer and activator for transcription (JAK/STAT) pathway, Ras mitogen activated protein

(MAPK) and the phosphatidylinositol 3 kinase (PI3/Akt) (Stahl, Boulton et al.

1994; Stahl, Farruggella et al. 1995; Oh, Fujio et al. 1998).

LIF mRNA has been found in both peripheral nervous system (PNS) and the CNS. LIF is expressed by Schwann cells, neurons and glial cells including astrocytes and microglia (Lemke, Gadient et al. 1996; Banner, Moayeri et al.

1997; Cheng and Patterson 1997; Dowsing, Morrison et al. 1999). Thus, LIF has pleiotropic effects ranging from the induction of cellular proliferation to the maintenance of cultured murine embryonic stem cell totipotentiality (Williams,

Hilton et al. 1988). Specifically in the CNS, LIF was shown to have both proliferative and protective effects on glial cells. LIF stimulated glial fibrillary acidic protein (GFAP) expression in astrocytic progenitors, whereas in OL

progenitor cultures, LIF enhanced myelin basic protein expression, survival, and

maturation (Mayer, Bhakoo et al. 1994; Nakagaito, Yoshida et al. 1995)

In disease states, LIF have been shown to attenuate the progression of

pathological disorders. LIF enhanced neuronal survival when placed at the site

of axotomized sensory and motor neurons (Oh, Fujio et al. 1998). LIF treatment

rescued OLs and reduced demyelination following spinal cord injury through

STAT 3 and PI3/Akt survival pathway activation (Metcalf and Gearing 1989;

Azari, Profyris et al. 2006). Furthermore, LIF administration attenuated demyelination and OL loss in experimental autoimmune encephalomyelitis

(EAE), the mouse model of multiple sclerosis, where the addition of anti-LIF

antibody exacerbated EAE disease pathology (Metcalf and Gearing 1989;

Metcalf and Gearing 1989; Butzkueven, Zhang et al. 2002). LIF rescued mature

cultured OLs from interferon γ and tumor necrosis factor α induced cell death,

through the activation of both JAK/STAT and PI3/Akt pathway (Slaets, Dumont et

al. 2008).

The release of pro-inflammatory cytokines following ischemic injury leads

to the expansion of the umbra, via activation of cellular cascades resulting in

macrophage activation and cell death. Therefore the discovery that LIF inhibits

the production of pro-inflammatory mediators including TNFα and ROS by

macrophages is relevant to the development of therapies against the progression

of stroke pathology (Hendriks, Slaets et al. 2008). Furthermore, endogenous LIF

peaked between 90 min and 24 hrs following MCAO in the peri ischemia area in

rats, a result that maybe explained as an intrinsic attempt by the brain to combat

the ischemic insult (Suzuki, Tanaka et al. 2000; Slevin, Krupinski et al. 2008). LIF

is selectively blood brain barrier permeable, thus, under pathological conditions

such as spinal cord injury, there is an up regulation of LIF transport across the

blood brain barrier and into the brain (Mayer, Bhakoo et al. 1994). This was

shown when animals were subjected to lipopolysaccharide (LPS) treatment and

an up regulation of LIF transport system at the blood brain barrier was identified

(Pan, Yu et al. 2008). In a subsequent study the site directed treatment of LIF to

the cerebral cortex adjacent to the necrotic area following MCAO significantly reduced infarct volume; this was more so a direct result of JAK stat activation and to a lesser extent PI3/Akt activation (Suzuki, Yamashita et al. 2005).

The promising effects of LIF obtained both in cell culture and animal model of different disease pathologies led to the development of a pharmacological recombinant LIF. The pharmacological form of LIF was developed to treat chemotherapy induced neuropathy. Phase 1 trials began in

1997 and a phase two trial is currently underway (Kurek 2000). Because of its pleiotropic activity, caution is placed on the usage of LIF as a treatment. In experiments where LIF was over expressed, animals showed weight loss, overgrowth of medullary bone tissue in long bones, calcification of the liver heart and skeletal muscle, thymus atrophy and the depletion of spermatogonia from males seminiferous tubules of the testes (Metcalf and Gearing 1989; Metcalf and

Gearing 1989). On the other hand, the deletion of LIF results in an array of abnormalities including abnormal development of the hippocampus, olfactory

receptor neurons and the inability of females to become pregnant (Escary,

Perreau et al. 1993; Bugga, Gadient et al. 1998; Moon, Yoo et al. 2002).

AKT Survival Pathway

HUCB cells secrete growth factors, chemokines, matrix metalloproteinase

inhibitors and interleukins (Newman, Willing et al. 2006; Neuhoff, Moers et al.

2007). Of these soluble factors, many including IGF1, VEGF and LIF are known

to promote cell survival via Akt phosphorylation/activation (Kulik, Klippel et al.

1997; Jin, Mao et al. 2000; Neuhoff, Moers et al. 2007). These results implicate the serine/threonine kinase Akt as a target of HUCB cell therapy. Upon phosphorylation, Akt is an integral part of the cellular machinery involved in survival. Phosphorylated Akt promotes survival by inhibiting the activation of apoptotic substrates while stabilizing substrates involved in survival pathways

(Kennedy, Wagner et al. 1997). Activated Akt phosphorylates Bcl-2-associated death promoter (BAD), pro caspase 9, Forkhead, and nuclear factor kappa B

(NFκb) (Datta, Brunet et al. 1999; Hanada, Feng et al. 2004; Zhao, Sapolsky et al. 2006).

When unphosphorylated, substrates such as BAD targets Bcl-2 in the mitochondria, causing cytochrome c release leading to caspase 9 cleavage

(Datta, Brunet et al. 1999; Zhao, Sapolsky et al. 2006). Caspase 9 is an initiator caspase which proteolytically cleaves execution pro caspase 3 and caspase 7

(Cryns and Yuan 1998; Datta, Brunet et al. 1999). Caspase 3 plays a principal role in the early apoptotic cascade leading to the cleavage of downstream

substrates such as poly (ADP-ribose) polymerase (PARP) (Nicholson, Ali et al.

1995; Le Rhun, Kirkland et al. 1998). PARP cleavage and NAD+ depletion as a

result of oxidative stress and DNA damage, leads to apoptosis inducing factor

(AIF) translocation to the cytoplasm from the mitochondria. AIF then migrates to

the nucleus where it induces chromatin lysis and cell death (Yu, Andrabi et al.

2006). Thus, phosphorylated Akt promotes survival by blocking the

aforementioned cell death cascades and stabilizing survival-associated

complexes through kinase activity (Kennedy, Wagner et al. 1997; Zhao, Sapolsky

et al. 2006).

Antioxidant Expression in the Brain

Delaying apoptotic cascades are necessary for inhibiting the progression of stroke pathology, but combating the production of ROS and RNS also plays a vital role. OL susceptibility to oxidative stress highlights an area that could be

exploited and prove beneficial to cellular homeostasis in the infarct boundary.

Thus, the identification and regulation of specific antioxidants in the cerebral

white matter post stroke could be a therapy defining focus. Two such

antioxidants are Mt3 and Prdx4. HUCB cells modulate both antioxidants

expression in the cerebral white matter OL (Rowe, Leonardo et al. 2010).

MT3

Metallothionein 3 (Mt3) is a small 68 amino acid protein that is extensively

involved in the storage and transport of metals such as copper. Mt3 is primarily

31 expressed in the CNS and maintains cellular homeostasis via detoxification of heavy metals such as mercury, and has also been shown to have antioxidant functions against ROS and RNS (Hidalgo, Aschner et al. 2001). In disease conditions, Mt3 is increased following stab wound in the cortex three days post injury and expression is returned to basal levels three weeks following experiment (Hozumi, Inuzuka et al. 1995; Hozumi, Inuzuka et al. 1996; Acarin,

Gonzalez et al. 1999). Using the stab wound model, Mt3 was shown to promoted tissue repair following injury (Hozumi, Uchida et al. 2006). In disease states such as Alzheimer’s (AD), Mt3 protein expression is significantly decreased. MT3 has been postulated to play a role in the accumulation of amyloid beta plaques due to the deregulation of zinc as a result of decreased Mt3 expression. Specifically, zinc is an active component of plaque formation, thus the accumulation of zinc in the absence of Mt3 is believed to exacerbate AD pathology (Uchida, Takio et al.

1991). A reduction in Mt3 was also observed post stroke. Following MCAO, Mt3 is gradual reduced until day 7, after which Mt3 gradually increased to basal levels by day 28 (Inuzuka, Hozumi et al. 1996). Therefore the maintenance or upregulation of Mt3 in affected tissue could play an active role in the attenuation of disease progression.

HUCB cell treatment 48 hrs post MCAO increased Mt3 protein expression and reduced apoptosis in the cerebral white matter (Rowe, Leonardo et al. 2010).

This protection points to Mt3 dual roles, activation of Akt survival pathway and antioxidant function. Mt3 binds to TrkA receptor tyrosine kinases to activate the

PI3K/Akt signaling pathway reducing H2O2 and doxorubicin (Dox) induced

caspase activation and cell death (Kim, Hwang et al. 2009). An additional study

showed that Mt3 protects against oxidative stress via Akt activation and

upregulating heme oxygenase 1, resulting in decreased ROS generation,

caspase 3 activation and neuronal cell death (Hwang, Kim et al. 2008). In these

experiments assessing Mt3 induced Akt activation, the use of Akt inhibitors completely abolished the protection conferred by Mt3 treatment (Hwang, Kim et al. 2008; Kim, Hwang et al. 2009). Mt3 attenuated glutamate neurotoxicity by quenching nitric oxide (NO) elicited by glutamate (Montoliu, Monfort et al. 2000).

Furthermore, Mt3 knockout animals showed increased susceptibility to kainic

acid whereas over expression of Mt3 reduced this susceptibility to kainic acid and

increased resistance to kainic acid (Erickson, Hollopeter et al. 1997). Mt3 has

also proven to rescue neurons by activating NFκb via the phosphorylation of IκBα

leading to IκBα degradation (Kim, Hwang et al. 2009). Thus, the regulation of Mt3

contributes to cellular detoxification and the activation of survival pathways

leading to cell survival in various disease states.

Prdx4

Peroxiredoxin (Prdx)4 is an additional antioxidant upregulated following

HUCB cell treatment in the external capsule of rats subjected to MCAO (Rowe,

Leonardo et al. 2010). Prdxs are biological antioxidants. This family of antioxidant

enzymes function through peroxidase activity reducing hydrogen peroxide,

peroxynitrite and other organic hydroperoxides (Hofmann, Hecht et al. 2002). Six

different Prdx have been classified to date; all have a common catalytic

mechanism of being unfolded, fully folded or a conserved conformation. Prdx4

mechanism involves the oxidation of a cysteine residue by the peroxide substrate causing a conformational change allowing the formation of a disulfide with the cysteine residue (Hall, Karplus et al. 2009). This action catalytically convert

substrates such as hydrogen peroxide to water. This activity detoxifies the

cellular environment, and the active enzyme is regenerated following reduction of

the disulfides (Zito, Melo et al. ; Hall, Karplus et al. 2009). This cycle is referred to

as the thiol/sulfenic acid redox cycle (Tanaka, Hijioka et al. 2004). There have

been at least six identified peroxiredoxins but only Prdx4 has been shown to

regulate thromboxane A2 receptor that regulates oxidative stress (Giguere,

Turcotte et al. 2007). Thromboxane A2 receptor is known to be upregulated by

oxidative stress and has been shown to contribute to cellular injury upon

activation during oxidative stress (Valentin, Field et al. 2004). Prdx4 also

undergoes structural changes in response to oxidation; this activity is thought to

prevent free radical induced aggregation of proteins (Jang, Lee et al. 2004; Kang,

Rhee et al. 2005). Oxidative stress leads to protein unfolding in the cytoplasm;

the chaperone activity of Prdx4 prevents the aggregation of these proteins,

where protein aggregation is known to facilitate disease pathology. In mouse, the

over expression of Prdx4 prevented p53 or epidermal growth factor induced

mitochondrial ROS production (Wong, Chun et al. 2000). The inhibition of ROS

production by Prdx4 over expression highlights the importance of this protein in

the prevention of ROS induced cellular dysfunction. The literature has shown the

importance of antioxidants in the brain following ischemic injury. Among these,

Prdx4 could play a vital role in the preservation of cells and reduction in stroke

induced morbidity and potential mortality.

HUCB Cells Therapy to Protect the White Matter OL

Presently, our laboratory is investigating HUCB cells as a potential treatment for stroke. In a report published by Hall. et al, our laboratory has

demonstrated the protective nature of HUCB cell treatment on OLs subjected to

OGD. Yet, the mechanisms by which HUCB cell rescue OLs remains to be established. Here we will examine how these cells protect OLs and evaluate the mechanism of action. These experiments are discussed herein.

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CHAPTER TWO

CORD BLOOD ADMINISTRATION INDUCES OLIGODENDROCYTE

SURVIVAL THROUGH ALTERATIONS IN GENE EXPRESSION

D.D. Rowe, MS, C.C. Leonardo, PhD, A.A. Hall, PhD,

M.D. Shahaduzzaman, MD,

L.A. Collier, BS, A.E. Willing, PhD, K.R. Pennypacker, PhD.

Abstract

Oligodendrocytes (OLs), the predominant cell type found in cerebral white matter, are essential for structural integrity and proper neural signaling. Very little is known concerning stroke-induced OL dysfunction. Our laboratory has

shown that infusion of human umbilical cord blood (HUCB) cells protects striatal

white matter tracts in vivo and directly protects mature primary OL cultures from

oxygen glucose deprivation (OGD). Microarray studies of RNA prepared from

OL cultures subjected to OGD and treated with HUCB cells showed an increase

in the expression of 33 genes associated with OL proliferation, survival, and

repair functions, such as myelination. The microarray results were verified using

quantitative RT-PCR for the following eight genes: U2AF homology motif kinase

1 (Uhmk1), insulin induce gene 1 (Insig1), metallothionein ( Mt3), tetraspanin 2

(Tspan2), peroxiredoxin 4 (Prdx4), stathmin-like 2 (Stmn2), myelin

oligodendrocyte glycoprotein (MOG), and versican (Vcan).

Immunohistochemistry showed that MOG, Prdx4, Uhmk1, Insig1 and Mt3 protein

expression were upregluated in the ipsilateral white matter tracts of rats infused

with HUCB cells 48 hrs after middle cerebral artery occlusion (MCAO).

Furthermore, promoter region analysis of these genes revealed common

transcription factor binding sites, providing insight into the shared signal

transduction pathways activated by HUCB cells to enhance transcription of these

genes. These results show expression of genes induced by HUCB cell therapy

that could confer oligoprotection from ischemia.

Keywords

Stroke; white matter; human umbilical cord blood cells; ischemia;

microarray; anti-oxidant

Abbreviations

OL, oligodendrocyte; HUCB, human umbilical cord blood; OGD, oxygen

glucose deprivation; Uhmk1, U2AF homology motif kinase 1; Insig1, insulin

induced gene 1; Mt3, metallothionein 2; Tspan2, tetraspanin 2; Prdx4,

peroxiredoxin 4; Stmn2, stathmin-like 2; MOG, myelin oligodendrocyte

glycoprotein; Vcan, versican; MCAO, middle cerebral artery occlusion; BDNF,

brain derived neurotrophic factor; NGF, nerve growth factor; GDNF, glial cell

derived neurotrophic factor; IGF-1, insulin like growth factor 1; DMEM, Dulbecco

modified eagle medium; PDGF-AA, platelet derived growth factor-AA; LDH, lactate dehydrogenase; GADPH, glyceraldehyde-3-phosphate dehydrogenase;

NG2, chondroitin sulfate proteoglycan; O4, Oligodendrocyte marker O4; MBP, myelin basic protein; GFAP, glial fibrillary acidic protein; RIP, Receptor interacting protein; EVI1, ecotropic viral integration site 1; MZF1, myeloid zinc finger protein; GATA1, GATA-binding factor 1; NK6.1, NK6 homeobox 1; PAX6, pax-6 paired domain binding site; Sox-5, SRY (sex determining region Y)-box 5;

SRF, serum response factor; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; IL-6, interleukin 6; H-I, hypoxic ischemia; AU, absorbance units.

Introduction

Stroke is the third leading cause of death in the United States, with

ischemic strokes accounting for 83% of all strokes (Lloyd-Jones, Adams et al.

2009). Ischemic brain injury affects both white and gray matter. Although white

matter integrity is essential to proper neuronal communication, much of current

research is focused exclusively on neuronal damage. Accounting for 50% of

brain volume in humans, white matter and the oligodendroglia that myelinate

these areas play an integral role in proper brain function (Miller, Alston et al.

1980). The myelin produced by OLs not only supports axonal structural integrity,

but is also essential in impulse integration (Baumann and Pham-Dinh 2001).

Thus, white matter protection is necessary to dampen stroke-induced injury and

its progressive pathology (Arai and Lo 2009).

In addition to myelination, OLs support the survival and function of neurons by regulating axonal size and ion channel clustering. OLs also secrete trophic factors such as BDNF, NGF, GDNF and IGF-1, all of which aid in cell

survival and maintenance (Baron-Van Evercooren, Olichon-Berthe et al. 1991;

Kaplan, Meyer-Franke et al. 1997; Wilkins, Majed et al. 2003; Noble 2005 ). Of

the different types of glia, OLs are the most vulnerable to hypoxic and

hypoglycemic conditions, yet the precise mechanisms underlying this

susceptibility are unknown (Lyons and Kettenmann 1998).

HUCB cell therapy is an emerging treatment for CNS injury. The

immaturity of HUCB cells contribute to the characteristic low immunogenicity

(Sanberg, Willing et al. 2005). HUCB cells are less immunogenic than other

stem cell treatments such as bone marrow and thus elicits lower

immunomodulatory effects (Sanberg, Willing et al. 2005; Wang, Yang et al.

2009). In vivo, HUCB cells migrate to the site of injury, resulting in reduced infarct volumes, neuroprotection and preservation of white matter following

MCAO (Vendrame, Cassady et al. 2004; Newman, Willing et al. 2005; Newcomb,

Ajmo et al. 2006; Hall, Guyer et al. 2009). Furthermore, multipotential stem cells

derived from HUCB cells secrete neuroprotective, angiogenic and anti-

inflammatory factors resulting in a functional recovery in spinal cord injuries

(Chua, Bielecki et al. 2010). In vitro experiments showed that in addition to

growth factors, HUCB cells secrete cytokines, matrix metalloproteinase inhibitors,

and interleukins (Neuhoff, Moers et al. 2007). Additionally, HUCB cells co-

incubated with OLs reduced OGD-induced apoptosis by decreasing activated

caspase 3 (Hall, Guyer et al. 2009). Despite these potent protective actions and

known soluble factors, the precise pathways involved in HUCB cell-mediated OL

survival have yet to be elucidated.

The present study examined changes in the gene expression profiles of

primary OL cultures subjected to OGD to elucidate the protective pathways

induced by co-incubation with HUCB cells. Microarray results revealed that 33

genes were significantly increased in OLs co-incubated with HUCB cells and

exposed to OGD. The upregulation of the following genes were confirmed by

qRT-PCR: Uhmk1, Insig1, Mt3, Tspan2, Prdx4, Stmn2, MOG, and Vcan gene

expression. Immunohistochemical analysis of tissues from rats treated with

HUCB cells 48 hrs after MCAO demonstrated increased protein expression of

Uhmk1, Insig1, Mt3, Tspan2, Prdx4, and MOG. Future experiments identifying

the mechanisms by which HUCB cells enhance the expression of protective

genes in OLs will provide insight into novel therapies to combat stroke-induced

white matter injury.

Results

Characterization of Mature OLs

Antibodies specific for NG2, O4 and MBP were utilized in double

immunofluorescence staining to determine OL developmental stage in vitro (Fig.

1). NG2 is a reliable marker throughout the course of OL differentiation in vitro,

while O4 is expressed by immature OLs. Furthermore, the expression of

myelinating proteins such as MBP denotes the mature OL phenotype. Six hours

following PDGF-AA withdrawal, NG2 and O4 colocalized in OLs with immature

morphology as indicated by the relatively low number of processes (Fig. 1A).

MBP was not detected at this time point. By 36 hrs following PDGF-AA removal, colocalization of NG2 and MBP (Fig. 1B) was evident in OL cultures. The prominent upregulation of MBP and the increased number of OL processes at 36 hrs signifies the progression of OLs to the mature phenotype that is present in the adult rat brain.

Secreted Factors from HUCB Cells Protect Mature OLs

Cell death is associated with LDH release through the plasma membrane, and therefore media levels of LDH were measured to assess OL injury after OGD

(Fig. 2). Media from OL cultures exposed to OGD showed significantly increased

LDH levels compared to that from normoxic controls (p < 0.01, n = 7).

Furthermore, HUCB cell treatment demonstrated oligoprotection. Media from OL

cultures co-incubated with HUCB cells and subjected to OGD showed

significantly reduced LDH levels relative to cultures subjected to OGD alone (p <

0.05, n = 7). Importantly, the fact that HUCB cells were separated from OLs by

transwell inserts indicates that HUCB cells exerted these protective effects

through the release of soluble factors rather than through direct cellular contact.

HUCB Cell Protection is Associated with Changes in Gene

Expression

Affymetrix microarray was utilized to detect changes in gene expression elicited by HUCB cells that were co-incubated with OLs during OGD. Of the 33 genes detected, eight genes encoding proteins associated with OL proliferation, survival, and repair functions were selected for further investigation (Table 1, bold font): Uhmk1, Insig1, Mt3, Tspan2, Prdx4, Stmn2, MOG, and Vcan. Genes listed in Table 1 exclude expressed sequence tags and exhibit fold changes ≥1.5

compared to OGD controls.

qRT-PCR Verification of Microarray Results

qRT-PCR was performed to validate gene expression data obtained by

microarray analysis. RNA was collected from supplementary experiments in

which HUCB cell co-incubation rescued OLs subjected to 24 hrs OGD. qRT-

PCR confirmed results obtained by microarray (Fig. 3). HUCB cell treatment

increased expression of all selected genes (Fig. 3A-H) when compared to OLs

subjected to OGD without HUCB cell treatment (p< 0.05). In addition, OGD

reduced the expression of Mt3, Tspan2, and Stmn2 (Fig. 3D-F) relative to normoxic controls (p< 0.05). HUCB cells also increased the gene expression of

MOG, Insig1, Prdx4, Mt3, Stmn2, and Vcan (Fig. 3A-D, F, H) under normoxic conditions when compared to normoxia only controls. Furthermore, a trend was also observed whereby HUCB cell treatment during OGD either maintained or increased mRNA expression levels relative to those of normoxic controls for all genes except Stmn2.

HUCB Cells Reduce Infarct Volume

Systemic administration of HUCB cells 48hrs post-stroke significantly reduced infarct volume. The fluorochrome Fluoro-Jade was used to detect degenerating neurons in coronal brain section taken from rats subjected to

MCAO and experimental groups that received HUCB cell treatment. HUCB cell treatment 48 hrs post-stroke significantly reduced infarct volume as compared to

MCAO only groups at 72 and 96hrs post stroke (*p<0.05, #p<0.01 respectively

(Fig.4). Sham operated groups were not significantly different from HUCB cell treated groups (p>0.05). Furthermore, brain tissues of HUCB cell treatment groups remained intact whereas tissue sections of MCAO only groups were fragile with signs of degradation.

HUCB Cells Protect OLs In Vivo

HUCB cells were administered 48 hrs post-stroke and sections were probed with anti-O4, a highly specific marker of OL cell bodies and processes

(Schachner, Kim et al. 1981; Sommer and Schachner 1982), to determine whether this therapy provided oligoprotection (Fig. 5). O4 immunoreactivity was ubiquitous throughout the ipsilateral external capsule in sections from animals treated with HUCB cells (Fig. 5A) and localized to cell bodies throughout the region (Fig. 5D). Sections from vehicle-treated and sham-operated rats also showed O4 immunoreactive cells, though they were sparsely distributed when compared to the ipsilateral hemisphere of HUCB cell treated rats (Fig. 5B,E).

Quantification of the percent area occupied by O4 immunoreactivity (Fig. 5C) showed that O4 was significantly increased in the ipsilateral hemisphere of animals that received HUCB cells relative to vehicle-treated and sham-operated controls (p<0.01).

HUCB Cells Induce Protein Expression

To expand on the microarray data from OL cultures, immunohistochemistry was performed to determine whether increased OL gene expression in vitro was consistent with increased gene product expression in vivo in the white matter rich region of the external capsule (Fig. 6 shows selected region). Experiments included sections from rats that were administered either vehicle or HUCB cells 48 hrs post-MCAO and rats that were subjected to sham-

MCAO and received vehicle injections. Immunostaining was performed for the

following proteins: Uhmk1 (Fig. 6), Prdx4, Mt3, MOG, Insig1, Tspan2, and Vcan

(Fig. 7). In general, sham-operated controls (Fig. 6E,F) showed nearly identical

staining patterns as vehicle-treated controls (Fig. 6C,D) with no apparent

differences in the expression of any proteins examined. While MOG (Fig. 7E,F)

and Vcan (Fig. 7K,L) immunoreactivity was present throughout the extracellular

space, immunoreactivity for all other proteins was restricted to cell bodies.

Quantification was performed by calculating the mean percent area for each

treatment group (Fig. 8). The contralateral hemisphere was utilized as an internal

control to adjust for ipsilateral brain swelling caused by edema. There was no

significant difference in the expression of any proteins when comparing sham-

operated and vehicle-treated controls. Uhmk1, Prdx4, Mt3, MOG and Insig1 were

upregulated in rats treated with HUCB cells compared to sham-operated and vehicle-treated controls (*p < 0.05, #p< 0.01), while Tspan2 and Vcan expression were unchanged.

Protein Expression and Localization

Double-label immunofluorescent staining was performed on sections from

animals subjected to MCAO to characterize the cellular expression profile of the

identified proteins. RIP, CD11b and GFAP were used for labeling of OL,

microglia and astrocytes, respectively, in conjunction with antibodies raised

against Prdx4, Mt3, Uhmk1, and Insig1. RIP colocalized with Prdx4, Mt3, Insig1,

and Uhmk1. RIP staining was localized in OL membranes as does Insig1, whereas, Prdx4 Mt3, and Uhmk1 labeled cytoplasmically (Fig 9). Although Prdx4

did not colocalize with CD11b (Fig. 10A-C), Prdx4-positive cell bodies colocalized

with GFAP-positive cells that exhibited the classic hypertrophic, stellate

morphology indicative of reactive astrocytes (Fig. 10D-F). Neither CD11b nor

GFAP colocalized with Mt3, Uhmk1 or Insig1 (Fig. 11).

Comparison of Gene Promoter

The promoter regions of genes upregulated in OLs co-cultured with HUCB

cells during OGD were explored by Genomatix software. Common transcription

factor binding sites were identified and included: EVI1, MZF1, GATA1, NK6.1,

PAX6, Sox-5, and SRF (Table. 2). These results suggest that the genes

identified by microarray are being transcriptionally elevated by similar signaling

pathways activated by the soluble factors secreted from the HUCB cells.

Discussion

The present study employed both in vitro and in vivo approaches to test

the efficacy of HUCB cells in reducing OL cell death and white matter injury,

respectively. LDH levels in media from OL cultures co-incubated with HUCB cells during OGD were reduced relative to OL-only cultures. As previously reported by

Newcomb et al 2006. and Vendrame et al 2004,. here we report that HUCB cell treatment 48 hrs post-stroke reduced infarct volume. Separate experiments showed that O4 immunoreactivity increased in the ipsilateral external capsule of rats treated with HUCB cells 48 hrs after MCAO. Upregulation of this OL marker was consistent with the previous report by Hall et al 2009. showing that HUCB

cell treatment increased MBP immunoreactivity. Thus, HUCB cells not only

protect OLs from OGD-induced injury in vitro, but also upregulate the expression of white matter-associated proteins after ischemia in vivo.

Based upon these data, further experiments were conducted to identify

the mechanisms by which HUBC cells confer protection. Gene expression

analysis of OL cultures subjected to OGD and treated with HUCB cells revealed

increased mRNA content of Uhmk1, MOG, Insig1, Mt3, Tspan2, Prdx4, Stmn2,

and Vcan. Additionally, the levels of Mt3, Prdx4, MOG, Insig1 and Uhmk1 gene

products were elevated in the ipsilateral external capsule of animals administered

HUCB cells 48 hrs after MCAO. Previous reports have demonstrated expression

of these proteins in OLs (Miyazaki, Asanuma et al. 2002; Jin, Lee et al. 2005;

Kursula 2008; Sim, Lang et al. 2008). Here, double-label immunohistochemistry

showed that the OL specific antibody RIP colocalizes with Prdx4, Mt3, Insig1 and

Uhmk1 whereas only Prdx4 colocalized with astrocytes, while none of the

proteins colocalized with microglia/macrophages. Both the increased gene

expression in culture and the lack of colocalization with other glial cell types in

vivo demonstrates that HUCB cells injected into MCAO rats caused OLs seated

within the cerebral white matter to upregulate these proteins.

To our knowledge, this is the first study linking upregulated expression of

genes and gene products with the protective effects of HUCB cell therapy within

the context of OL susceptibility and white matter injury resulting from ischemia.

HUCB cell-induced upregulations in Prdx4 and Mt3 observed here are consistent with the notion that HUCB cells provide protection to white matter by inducing

OLs to express proteins that combat oxidative damage. Oxidative stress is a

major cause of OL cell death resulting from OGD (Dewar, Underhill et al. 2003).

The Prdx family of anti-oxidants exerts protective effects through peroxidase

activity, detoxifying a range of free radical-forming organic hydroperoxides

(Hofmann, Hecht et al. 2002). In particular, Prdx4 regulates the thromboxane A2 receptor, a receptor which is upregulated by oxidative stress and contribute to

oxidative injury upon activation (Valentin, Field et al. 2004). Previous work

showed that thromboxane A2 expression was inhibited during oxidative stress by

Prdx4 over-expression (Giguere, Turcotte et al. 2007). In addition, the Prdx

family has also been shown to undergo structural changes to engage in

chaperone activity in response to excessive oxidation (Jang, Lee et al. 2004).

This chaperone activity may be a necessary function in the recovery of

oxidatively damaged cells by preventing free radical-induced aggregation of

cytosolic proteins (Jang, Lee et al. 2004; Kang, Rhee et al. 2005).

Similarly, the antioxidant Mt3 exerts its effects through metal detoxification

and free radical scavenging activity (Hozumi, Inuzuka et al. 1998; Uchida, Gomi

et al. 2002; Hwang, Kim et al. 2008). These mechanisms are of particular

relevance to the present study since iron is not only a critical co-factor in myelin

production, but is also highly reactive and can contribute to free radical formation

and lipid peroxidation (Braughler, Duncan et al. 1986; Connor and Menzies

1996). Additionally, OLs possess low concentrations of the antioxidant

glutathione, and oxidative stress leads to increased iron-mediated production of

ROS (Juurlink 1997; Juurlink, Thorburne et al. 1998). Thus, the protective effects

of HUCB cells likely result, at least in part, from the secretion of a factor or

factors that ultimately increase the expression of Mt3.

HUCB cell therapy has previously been shown to target the Akt signaling

pathway, as Akt inhibition diminishes the protective effects of HUCB cells

(Dasari, Veeravalli et al. 2008). Importantly, growth factors such as VEGF and interleukins such as IL-6, which are secreted by HUCB cells, have also been shown to activate Akt, leading to cell migration, angiogenesis, and cell survival

(Morales-Ruiz, Fulton et al. 2000; Six, Kureishi et al. 2002; Neuhoff, Moers et al.

2007; Wegiel, Bjartell et al. 2008). The present study identified several common transcription factor binding sites within the promoter regions of the genes identified by microarray. In particular, EVI1, MZF1, and GATA1 transcription occur downstream of PI3k/Akt activation (Yu, Chiang et al. 2005; Liu, Chen et al.

2006; Moeenrezakhanlou, Shephard et al. 2008), providing additional evidence that Akt is an important upstream activator responsible for the oligoprotective, anti-oxidant effects of HUCB cells. Taken together, these data show that HUCB cells release factors that transduce signaling converging on Akt, thereby increasing the transcription of oligoprotective genes.

In addition to combating oxidative stress, data here show that HUCB cell treatment alters the expression of proteins involved in microtubule regulation.

The concerted actions of MOG and Stmn2 inhibit microtubule polymerization, and Stmn2 was previously found to increase neurite outgrowth via this mechanism (Riederer, Pellier et al. 1997; Johns and Bernard 1999; Chiellini,

Grenningloh et al. 2008). Thus, these data provide evidence that upregulated

expression of MOG and/or Stmn2 acts to inhibit microtubule polymerization in

OLs, thereby increasing proliferation and/or migration and enhancing white

matter repair.

Indeed, previous findings showed that mature OLs retain the ability to

proliferate following injury (Wood and Bunge 1991). In addition to regulating microtubule dynamics through the phosphorylation of Stmn2 (Belmont and

Mitchison 1996), Uhmk1 induces proliferation and cell cycle progression through the phosphorylation of p27kip1(Nakamura, Okinaka et al. 2008). Interestingly,

Uhmk1 was also upregulated after MCAO in HUCB cell-treated rats. Here, the observed elevations in both Uhmk1 and O4 expression support the notion that

HUCB cell therapy protects white matter injury by inducing OL proliferation via

this pathway.

HUCB cell therapy may also alleviate white matter injury through

replacement of important somatic and/or axonal membrane lipids that are

degraded in response to H-I injury. The OL axon sheath is rich in

glycosphingolipids and cholesterol (Simons and Trajkovic 2006). Insig1 is

degraded when cholesterol is depleted within a cell (Gong, Lee et al. 2006), and

hypoxia increases Insig1 expression through a mechanism mediated by hypoxia

inducible factor 1α (Nguyen, McDonald et al. 2007). Thus, the elevations in

Insig1 likely reflect HUCB cell induction of cholesterol biosynthesis aimed at

remyelination or restoration of the cell membrane.

Although a role for HUCB cells in remyelination is also supported here by

increased O4 expression, future experiments are necessary to determine

precisely which proteins are associated with restoration of the myelin sheath or cell membrane viability in general. For example, Tspan2 is integrated into the myelin sheath membrane following active myelination, while Vcan is involved in

OL migration, proliferation, and structural integrity (Birling, Tait et al. 1999;

Sheng, Wang et al. 2005). Tspan2 and Vcan mRNAs were upregulated in OL cultures subjected to OGD and treated with HUCB cells, yet there was no significant difference in protein expression in vivo after MCAO. These data suggest that although Vcan and Tspan may be capable of enhancing axonal and/or plasma membrane viability in culture, the complex microenvironment present in the stroked brain determines which genes are translated and trafficked accordingly. Likewise, these differences in in vitro transcription and in vivo translation highlight the importance of combining multiple approaches to elucidate the protective pathways elicited by HUCB cells. Future studies investigating these pathways will provide insight into the precise factors secreted by HUCB cells that protect OLs and cerebral white matter following ischemia.

Experimental Procedure

Animal Care

All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals with a protocol approved by the

Institutional Animal Care and Use Committee at the University of South Florida.

Experiments were designed to minimize the number of animals required.

Sprague-Dawley rats were purchased from Harlan Labs (Indianapolis, IN),

maintained on a 12 hr light/dark cycle (7 am – 7 pm) in a climate-controlled room, and allowed access to food and water ad libitum. Neonatal rats birthed from untimed-pregnant dams were used for in vitro experiments and 300-350 g male rats were used for in vivo experiments. Measures taken to minimize pain and discomfort are described in the subsequent methodology.

Mixed Glial Culture Preparation

Postnatal day 3 rat pups were decapitated, brains removed, and meninges dissected away. Rat cortices were dissociated in a solution of 0.25% trypsin/2.21 mM EDTA, triturated, and pelleted. The pellet was re-suspended in DMEM

(Mediatech, Manassas, VA) supplemented with 2.5% fetal bovine serum, 10% horse serum, and 1% antibiotic/antimycotic (DMEM+). Trypan Blue exclusion was used to assess cell viability. Cells were seeded (1.5 x 107) into poly-L- lysine-treated 75 cm2 tissue culture flasks. Media was changed with fresh

DMEM+ the following day and cultures were incubated for 8 days at 37°C

(Gottschall, Yu et al. 1995).

OL Culture Purification

Mixed glial cultures were mechanically shaken for 1 hr to separate microglial cells from the OL/astrocyte monolayer and media was discarded.

astrocyte monolayer. The media was removed, the cells were pelleted and re-

suspended in DMEM+. Viable cells were then counted using Trypan Blue

exclusion. Microglia- and OL-containing media was added to 10 cm plastic tissue culture dishes at a density of 107 cells/dish and incubated for 15 min at

37°C (procedure repeated 3 times for microglial adherence to the plastic). After

incubation, the dishes were gently swirled and media collected. The remaining

suspension was pelleted, re-suspended in DMEM+, and plated on glass poly-L-

lysine-treated coverslips at 3 x 105 cells/coverslip (McCarthy and de Vellis 1980).

The following day, media was changed to Neurobasal complete (Neurobasal

supplemented with B-27, L-glutamine 0.5mM, and 10ng/ml PDGF AA) (Barres,

Schmid et al. 1993; Yang, Watanabe et al. 2005). OLs remained in Neurobasal

complete and PDGF-AA for 7 days to encourage proliferation. After the

proliferation period, PDGF-AA was withdrawn for 5 days to induce OL

differentiation into the mature phenotype (Yang, Watanabe et al. 2005).

Experiments were conducted immediately following the 5 day PDGF-AA

withdrawal. All in vitro experiments were conducted using > 95% pure OL

cultures, as previously described in (Hall, Guyer et al. 2009).

Oxygen Glucose Deprivation

OLs were seeded onto glass coverslips and randomly assigned to one of

two conditions: OGD (DMEM without glucose) or normoxia (DMEM with

glucose). Transwell inserts (0.2 µm: Nalge Nunc International, Rochester, NY)

were added to 6-well plates containing coverslips. The inserts provided a barrier

that prevented OL-HUCB cell contact but was permeable to media and soluble factors. Cryopreserved HUCB cells (ALLCELLS, Emeryville, CA) were rapidly thawed, washed, pelleted to remove the cryopreservatives and re-suspended in

10 ml DMEM with glucose and DNase (Sigma-Aldrich, St. Louis, MO; 50 kunitz units/ml). HUCB cells were seeded onto tissue culture inserts (1x105 cells/insert) and placed into the wells containing OL coverslips immediately prior to OGD exposure. Experimental groups not subjected to HUCB cell treatment received inserts containing an equal volume of DNase-supplemented DMEM with glucose.

A negative control of media alone and wells containing 1x105 HUCB cells with

DNase were included as controls to quantify HUCB cell contribution to the LDH assay for each experimental condition.

Cells undergoing OGD were placed in an air-tight hypoxica chamber. The chamber was then flushed with hypoxic gas (95% N2, 4% CO2, 1% O2; Airgas,

Tampa, FL) for 15 min and sealed for the duration of exposure. Normoxic cells were maintained in a standard tissue culture incubator. Cultures were subjected to OGD or normoxia for 24 hrs at 37°C. The media from each well was collected, clarified by centrifugation, and LDH analysis was performed immediately.

LDH Assay

OL cell death in culture was determined using the LDH assay (Takara Bio,

RNA Collection and Purification

All collection and purification steps were performed under nuclease-free conditions using DNAse/RNAse-free materials. For RNA lysate collection, 10 µl of β-Mercaptoethanol (Pharmacia Biotech, Uppsala, Sweden) was added to 1 ml

RTL buffer (Qiagen Inc., Valencia, CA) and 350µl of the resulting mixture was added to each OL-containing well to lyse the cells. Cell lysates were then collected and stored at -80°C prior to extracting the RNA. Qiagen’s RNeasy Mini

Kit was used to extract total RNA from each cell lysate using the optional Qiagen

RNase-Free DNase set for DNase digestion (Qiagen Inc). Following the extraction, 1µl of each RNA sample was tested in an Agilent 2100 Bio-analyzer to determine the purity and quantity of RNA present. The remaining sample was stored at -80°C for subsequent use with gene array.

Gene Array

Gene array was performed by the H. Lee. Moffitt Cancer Center

Microarray Core Facility utilizing a GeneChip 3000 Scanner, GCOS 1.4 with an

Affymetrix MAS 5.0 algorithm to generate signal intensities, and GeneChip Rat

Genome 230 2.0 Array (Affymetrix inc, Santa Clara, CA). Microarray data were

normalized to RNA from cultures exposed to OGD. Only genes with≥ 1.5 fold

increase and a signal intensity of > 100 were selected for further investigation.

For investigated treatment groups, samples were pooled (n=5) to obtain the

necessary RNA quantity and quality to perform this procedure.

Quantitative Real Time Polymerase Chain Reaction

Primers were ordered for selected sequences in which expression of

genes deemed vital to OL survival and proliferation were increased at least 1.5

fold after HUCB cell treatment. Uhmk1, Insig1, Mt3, Tspan2, Prdx4, Stmn2, and

MOG were purchased from SABiosciences (Frederick, MD; sequences are

proprietary). Vcan (Integrated DNA Technologies Coralville, IA) was examined

using the following primers:

Reverse 5’ TTT TAG GCA TTG CCC ATC TC

Forward 5’ ATG ACG TCC CCT GCA ACT AC

Total RNA (10 ng/µl) from OL cultures were subjected to qRT-PCR. The

RT reaction mixture consisted of 3µl Oligo (dT) Primers, 10 µl cDNA Synthesis

Master Mix (2X), 1 µl of Affinity Script RT/RNase Block enzyme mixture, and

RNase-free H2O to a total volume of 20 µl (Stratagene, La Jolla, CA). The reaction was incubated at 25°C for 5 min to allow primer annealing, then incubated at 42°C for 45 min to allow cDNA synthesis followed by 5 min incubation at 95°C to terminate the cDNA synthesis reaction.

Complementary DNA from the RT reaction was added to a PCR reaction

mix consisting of 1 µl cDNA, 12.5 µl 2X Brilliant 490 SYBR Green QPCR Master

Mix (Stratagene), 2 µl primer, and nuclease-free PCR grade H2O to a total volume of 25 µl. The samples were amplified using a BioRad ICycler (Bio-Rad

Laboratories, Hercules, CA) with the following protocol: heating to 95°C for 15 min followed by 40 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 55

°C, and 30 sec of elongation at 72°C. GADPH was selected as a reference gene and was used to calculate the mean normalized expression.

Determination of Promoter Response Elements

Accession numbers of OL genes shown by microarray and confirmed by qRT- PCR to increase expression were entered into Genomatix software

(ElDorado/Gene2Promoter v4.7.0;Genomatix Software Inc, Ann Arbor, MI). The promoter regions of selected genes were investigated for common transcription factor binding sites. Transcription factor families were determined and transcription factor binding sites conserved across the promoter regions of all selected genes were identified.

Laser Doppler Blood Flow Measurement

Prior to MCAO surgery, animals were anesthetized with 5% isofluorane/O2 in an induction chamber. Rats were treated prophylactically with Ketoprofen (10 mg/kg s.c.), atropine (0.25 mg/kg s.c.) and Baytril (20 mg/kg i.m.) in accordance with IACUC guidelines. Ketoprofen injections were continued 3 days post-MCAO to minimize pain and discomfort. A constant flow of anesthesia was supplied with an interfaced scavenging system (3-4% isofluorane, flow rate 1 L/min)

throughout the procedure. For Doppler insertion, the head was shaved and an incision was made lateral to the midline of the dorsal plates of the skull. The skin was spread and tissue covering the skull bone was pushed aside with a cotton- tipped applicator. Using a micro-drill, a small hole was drilled into the skull at 1 mm posterior and 4 mm lateral to bregma. A hollow stainless steel guide screw was positioned into the hole and a fiber optic filament (500 µm) was inserted through the screw guide and secured with Vetbond (3M, St. Paul, MN). Blood perfusion in the brain was monitored using the Moor Instruments (Devon,

England) Ltd laser Doppler with Moor LAB proprietary Windows-based software.

When surgery was complete, the screw guide was removed, bone wax placed in the burr hole and the scalp incision was sutured. Rats that did not show ≥ 60% reduction in blood perfusion during MCAO were excluded from the study (Hall,

Guyer et al. 2009)

MCAO and HUCB Cell Treatment

MCAO surgeries were performed as previously described (Butler, Kassed et al. 2002; Vendrame, Cassady et al. 2004; Hall, Guyer et al. 2009). Following implantation of the Doppler probe, the external carotid artery was exposed and isolated from the vagus nerve using blunt dissection. The artery was then ligated and transected near the bifurcation of the internal and external carotid arteries.

The stump of the external carotid was then used as a guide to advance a monofilament through the internal carotid to the origin of the middle cerebral artery. The filament was then sutured secure and the incision closed. For sham

surgeries, the Doppler probe was inserted and the external carotid exposed, but no filament inserted.

To determine whether HUCB cells were protective against stroke-induced injury, rats were injected (i.v, penile vein) with either HUCB cells (1x106 HUCB cells in 500 PBS (pH 7.4 + DNase) or vehicle (500 μL PBS + DNase only) 48 hrs after MCAO surgery. Sham-operated animals also received vehicle. Animals were then sacrificed 54, 72 and 96 hrs post-stroke and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde in PBS. The brains were removed and saturated with 4% paraformaldehyde in PB followed by increasing concentrations of sucrose in PBS (20%, 30%). Brains were then sectioned at 30

μm on a cryostat to include bregma 1.7mm through bregma -3.3, thaw mounted onto slides and stored at -20°C.

Fluoro-Jade Histochemistry

Brain sections were thawed, dried and rehydrated with 100% EtOH for 3 min, 70% EtOH for 1min, and 1min in ddH2O. Using a 0.06% KMnO4 solution, sections were oxidized for 15 min. After 3x1min washes in ddh20, brain sections were placed in a 0.001% solution of Fluoro-Jade (Histochem, Jefferson, AR) in

0.1% acetic acid for 30 min. Following incubation, sections were washed 4x3min in ddH2O, dried, cleared in xylene, and cover-slipped with DPX mounting medium

(VWR International Ltd, Poole, England).

Immunohistochemistry

For peroxidase detection, brain tissue sections were washed with PBS for

5 min and incubated in 3% hydrogen peroxide for 20 min. Sections were then

washed 3 times in PBS, incubated for 1 hr in permeabilization buffer (2% serum,

0.3% Triton X-100 and 0.3% 1M lysine in PBS) and incubated overnight at ˚C4

with primary antibody in antibody solution (2% goat serum, 0.3% Triton X-100 in

PBS). The following day, sections were washed with PBS and incubated 1 hr at

room temperature with secondary antibody in antibody solution (2% serum, 0.3%

Triton X-100 in PBS). Sections were then washed in PBS, incubated in Avidin-

Biotin Complex (ABC; Vector Laboratories Inc, Burlingame, Ca) mixture for 1hr,

washed again and visualized using a DAB/peroxide solution (Vector Laboratories

Inc). After 3 final washes, sections were dried, dehydrated with increasing

concentrations of EtOH (70%, 95%, 100%), cleared with xylene and cover-

slipped with DPX. Antibodies consisted of the following: mouse anti-MOG

(Abcam, Cambridge, MA; 1:250), rabbit anti-Uhmk1 (Protein tech group, Chicago

IL; 1:50), rabbit anti-Prdx4 (Abcam; 1:250), goat anti-Vcan (Santa Cruz

Biotechnology Inc, Santa Cruz, CA; 1:50), rabbit anti-Tspn2 (Sigma-Aldrich), goat anti-Insig1 (Santa Cruz Biotechnology Inc; 1:50), and rabbit anti-Mt3 (Sigma-

Aldrich; 1:50). Secondary detection was achieved using biotinylated secondary antibodies (Vector Laboratories; 1:300) corresponding to the respective species of primary antibodies.

For fluorescent labeling, tissue sections and cultured OLs were subjected to the same method used for peroxidase detection, prior to the secondary

antibody incubation, except that fluorescence samples were not incubated in

hydrogen peroxide. Double-label immunohistochemistry was achieved by co-

incubating the tissues or cells with primary antibodies raised in two distinct

species, followed by co-incubation with secondary antibodies conjugated to

distinct fluorophores. Following secondary antibody incubation, sections were

washed and cover-slipped using VectaShield Hard Set with DAPI (Vector

Laboratories). Antibodies used for fluorescent detection consisted of the

following: mouse anti-RIP (Millipore, Temecula, CA; 1:5000), rabbit anti-Prdx4

(Abcam; 1:500), mouse anti-O4 (Chemicon, Temecula, CA; 1:1000), mouse anti-

OX-42 (AbD Serotec, Kidlington, Oxford, UK; 1:1000), rat anti-MBP (Abcam;

1:1000), rabbit anti-NG2 (Chemicon; 1:500), rabbit anti-Uhmk1 (Protein tech

group; 1:50), goat anti-Insig1 (Santa Cruz Biotechnology Inc; 1:50), rabbit anti-

Mt3 (Sigma-Aldrich; 1:50), and mouse anti-GFAP (Chemicon; 1:1000).

Secondary antibodies used were Alexa Fluor 488 and 594 (Molecular Probes,

Eugene, OR; 1:1000). Negative controls were labeled in the absence of primary antibody corresponding with respective secondary, as discussed previously.

Image Analyses

For in vivo image analyses, brain sections from≥ 3 animals per group were used. Coronal brain sections encompassing the striatum (Bregma coordinates +1.7 through -0.3) were taken from each animal. Images were generated using a Zeiss Axioskop2 microscope controlled by Openlab

(Improvision Ltd, Lexington, MA) software. Images were captured with a Zeiss

Axiocam Color camera. The ImageJ 1.410 program (National Institutes of

Health, USA) was used to measure relative total intensity ratios of ipsilateral vs. contralateral hemispheres. Ratios were calculated for each animal due to ipsilateral brain swelling caused by edema. The group mean total intensity ratio for each experimental treatment group was used for comparisons across treatments. Total intensity analysis was conducted where treatment groups were blinded.

Statistical Analyses

Data from all experiments were quantified and analyzed using GraphPad

Prism 4.0 (GraphPad Software, La Jola, CA) software. Main effects were determined using one-way ANOVAs, followed by Dunnett’s post hoc tests to detect significant differences across treatment groups. When two variables were present, two-way ANOVAs were used followed by Bonferroni post hoc tests. A

“p” value < 0.05 was used as the threshold for significant differences.

Acknowledgements

This work was supported in part by the National Institutes of Health (R01

NS052839), the American Heart Association (0715096B to A.A.H.), and the

University of South Florida Department of Molecular Pharmacology and

Physiology. The authors also thank Dr. Javier Cuevas and the H. Lee. Moffitt cancer center Microarray core for their contributions.

Disclosure

A.E. Willing is a consultant to Saneron CCEL Therapeutics, Inc. and is an inventor on cord blood related patents.

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Figure 1. OLs Differentiate Into the Mature Phenotype. Photomicrographs show immunofluorescent staining of OL cultures at selected time points following PDGF-AA withdrawal. (A) 6 hrs after withdrawal, NG2 (red) and O4 (green) colocalized in OLs that exhibited both bipolar and immature morphology, as indicated by the lateralized orientation of processes and the relatively low number of processes, respectively. (B) At 36 hrs, NG2-positive OLs (red) expressed MBP (green) and contained greater numbers of processes, indicating that this withdrawal period was sufficient for differentiation into the mature phenotype. Scale bars = 50μm.

Figure 2. HUCB Cells Decrease LDH Release From OLs Subjected to 24 hrs OGD. Media from OL cultures subjected to OGD-only contained elevated levels of LDH compared to media from normoxic controls, demonstrating OGD-induced cellular injury. OL cultures subjected to OGD were rescued by co-incubation with HUCB cells, as LDH release was reduced back to levels of normoxic controls (*p < 0.01, n=7).

Figure 3. Affymetrix Gene Array Fold Changes are Confirmed by qRT-PCR. HUCB cell treatment of OLs exposed to 24 hrs OGD significantly increased gene expression of MOG, Insig1, Prdx4, Mt3, Tspan2, Stmn2, Uhmk1 and Vcan (A-H) as compared to OLs subjected to OGD alone (* p < 0.05, n = 5). Additionally, HUCB cell treatment of OLs exposed to normoxia increased the expression of MOG (A), Insig1 (B), Prdx4 (C), Stmn2 (F), and Vcan (H) as compared to non- treated normoxic controls (^ p < 0.05 n = 5). Under OGD conditions, OL expression of Mt3 (D), Tspan2 (E) and Stmn2 (F) were significantly reduced in non-treated cells compared to both normoxic groups (# p < 0.05, n = 5).

Figure 4. HUCB Cells Reduce Infarct Volume. HUCB cells provide neuroprotection when given systemically 48hrs post-stroke. Photomicrographs depict Fluoro-Jade staining of coronal rat brain sections at time points 54, 72 and 96 hrs post-MCAO. Infarct volume remained constant in MCAO only groups at 54 hrs (A), 72 hrs (B), and 96 hrs (C) post MCAO. Whereas HUCB cell administration reduced infarct volume at 72 hrs (E) and 96 hrs (F) post-stroke (* p < 0.05, # p < 0.01, respectively n = 4) while not significantly different from sham operated animals (G-I) (p > 0.05) at respective time points. Bar graph (G) shows the percent volume quantification of the ipsilateral (stroked) hemisphere compared to the contralateral (non-stroked) hemisphere for each group.

Figure 5. HUCB Cells Rescue OLs of the External Capsule Following Ischemic Insult. O4 immunoreactivity was abundant throughout the ipsilateral external capsule of animals treated with HUCB cells 48 hrs post-MCAO (A, D). Vehicle (B) and sham-operated (E) controls also expressed O4, though immunoreactivity was sparsely distributed and less prominent compared to HUCB cell-treated animals. Quantification showed that HUCB cell treatment significantly increased O4 immunoreactivity relative to both vehicle-treated and sham-operated controls (* p < 0.01, n = 3). Scales bar = 50 μm. Arrows points to O4 positive staining.

Figure 6. HUCB Cells Increase White Matter Uhmk1 Expression Following Ischemic Insult. HUCB cell treatment (A,B) 48 hrs post-MCAO significantly increased Uhmk1 expression in the ipsilateral hemisphere of the external capsule compared to vehicle (C,D) and sham-operated (E, F) controls (* p < 0.05, n = 3). Low magnification scale bars = 100 μm; high magnification inset scale bars = 20 μm.

Figure 7. HUCB Cells Alter White Matter Protein Expression Following Ischemic Insult. Photomicrographs show increased expression of Prdx4 (A), Mt3 (C), MOG (E) and Insig1 (G) in the ipsilateral hemisphere of animals treated with HUCB cells 48 hrs post-MCAO compared to vehicle-treated controls (B,D,F,H, respectively). No differences were observed in the expression of Tspan (I,J) or Vcan (K,L) in response to HUCB cell treatment. Scale bars = 50 μm. Arrows points to positive staining.

Figure 8. Immunohistochemical Quantification of White Matter Protein Expression. HUCB cell treatment 48 hrs post-MCAO resulted in increased expression of Uhmk1 (A), Prdx4 (B), Mt3 (C), MOG (D), and Insig1 (E) in the ipsilateral external capsule compared to vehicle-treated and sham-operated controls (*p < 0.05, #p < 0.01 n = 3). No significant differences were detected for Tspan2 (F) or Vcan (G).

Figure 9. Prdx4, Uhmk1, Insig1 and Mt3 Colocalized With OL Marker RIP. Photomicrographs depicts immunoflourescent double-labeling of OL specific antibody RIP (A, D, G, J) and antibodies generated against Prdx4 (B), Mt3 (K), Insig1 (E), and Uhmk1 (H). RIP and Insig1 are colocalized (F) in OL membranes, whereas Prdx4 (C), Mt3 (L), and Uhmk1 (I) are cytoplasmically localized. Scale bars = 50 μm. Arrows points to positive staining.

Figure 10. Prdx4 is Expressed in Astrocytes but not Microglia/macrophages Following Ischemic Insult. Double-label Immunohistochemistry for Prdx4 (A) and CD11b (B) shows that Prdx4 is not expressed in CD11b-positive microglia/macrophages (C) contained within the ipsilateral external capsule. Prdx4 (E) and GFAP (D) colocalization shows astrocytic expression of Prdx4 (F) within the white matter following ischemic insult. Scale bars = 100 μm.

Figure 11. Mt3, Uhmk, and Insig1 are not Expressed in Microglia/macrophages or Astrocytes Following Ischemic Insult. Immunofluorescent double-labeling shows that while expression is evident in the ipsilateral external capsule following MCAO, Mt3, Uhmk1, and Insig1 did not colocalize with CD11b-positive microglia/macrophages (A, C, E) or GFAP- positive astrocytes (B, D, F). Scale bars = 50 μm.

Table 1. HUCB Cell Treatment Alters Gene Expression in OLs Subjected to 24 hrs OGD. Table shows OL genes for which HUCB cell treatment during OGD caused a fold change ≥ 1.5 compared to non-treated OGD controls. Genes were grouped based on functional relevance, and those associated with OL survival, proliferation, and myelination (bold font) were selected for further investigation.

Table 2. Common Transcription Factor Binding Sites Present in the Promoters of Upregluated Genes. Table shows common transcription factor binding sites identified in the promoter regions of Prdx4, Mt3, Insig1, MOG, Uhmk1, Tspan2, Vcan and Stmn2. 8/8 denotes transcription factor binding sites present in all 8 genes, whereas 7/8 denotes transcription factor binding sites present in 7 of the 8 selected genes.

CHAPTER THREE

HUMAN UMBILICAL CORD BLOOD CELLS PROTECT

OLIGODENDROCYTES FROM BRAIN ISCHEMIA THROUGH AKT SIGNAL

TRANSDUCTION

D.D. Rowe, MS, C.C. Leonardo, PhD, L.A. Collier, BS, A.E. Willing, PhD,

K.R. Pennypacker PhD.

Abstract

Human umbilical cord blood (HUCB) cells protect against ischemic injury,

yet the mechanism of protection remains unclear. This study examined the role

of Akt activation in HUCB cell-mediated protection of oligodendrocytes (OLs). We

have employed the use of in vitro and in vivo models of ischemia to evaluate Akt activation following HUCB cell treatment. As previously reported, HUCB cells rescued cultured mature OLs from oxygen glucose deprivation (OGD) induced cell death, as measured by lactate dehydrogenase (LDH) assay.

Immunohistochemical analysis showed that HUCB cell treatment enhanced Akt phosphorylation in cultured OLs subjected to 24 hrs OGD. The addition of Akt

Inhibitor IV to cultured OLs abolished the protective effects of HUCB cells and their induction of the antioxidant enzyme peroxiredoxin 4 (Prdx4). Systemic administration of HUCB cells 48 hrs post middle cerebral artery occlusion

(MCAO) activated the Akt kinase through phosphorylation at serine 473, while reducing proteolytic cleavage of caspase 3 in the ipsilateral external capsule.

These results demonstrate that Akt activation is an integral component of HUCB cell mediated protection from ischemic injury.

Keywords

Stroke; white matter; human umbilical cord blood cells; ischemia; antioxidant; Akt activation, Caspase 3 activation.

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Abbreviations

HUCB, human umbilical cord blood; OL, oligodendrocyte; OGD, oxygen

glucose deprivation; LDH, lactate dehydrogenase; Prdx4, peroxiredoxin 4;

MCAO, middle cerebral artery occlusion; DMEM, dulbecco modified eagle

medium; PDGF-AA, platelet derived growth factor-AA; DMSO, dimethyl sulfoxide;

GADPH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate buffered saline; RIP, Receptor interacting protein; Mt3, metallothionein 3; GDNF, glial cell derived neurotrophic factor; BDNF, brain derived neurotrophic factor; LIF, leukemia inhibitory factor; IL-1β, interleukin-1 beta; IL-6, interleukin-6; IGF1, insulin like growth factor 1; VEGF, vascular endothelial growth factor; TIMP-1,

TIMP metallopeptidase inhibitor 1; BAD, bcl2 associated death promoter; PARP,

poly (ADP-ribose) polymerase; ROS, reactive oxygen species; RNS, reactive

nitrogen species.

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Introduction

Until recently, oligoprotection has been largely overlooked in the development of new approaches to treat cerebral ischemia. Human umbilical cord blood (HUCB) cells have been highly efficacious in treating experimental stroke. HUCB cells rescued oligodendrocytes (OL)s subjected to oxygen glucose deprivation (OGD) by increasing survival associated and antioxidant gene expression while inhibiting apoptotic activators (Rowe, Leonardo et al. ; Hall,

Guyer et al. 2009). Moreover, HUCB cells reduced infarct volume, promoted behavioral recovery and preserved white matter bundles following middle cerebral artery occlusion (MCAO) (Vendrame, Cassady et al. 2004; Newman,

Willing et al. 2005; Hall, Guyer et al. 2009).

HUCB cells are comprised of proliferative hemopoietic colony-forming, endothelial precursor and mesenchymal progenitor cells (Nakahata and Ogawa

1982; Nieda, Nicol et al. 1997; Erices, Conget et al. 2000). The protective properties of this therapy have been attributed to the soluble factors secreted by these cells, many of which are known activators of the Akt signal transduction pathway (Kulik, Klippel et al. 1997; Jin, Mao et al. 2000; Newman, Willing et al.

2006; Neuhoff, Moers et al. 2007). Upon phosphorylation at serine 473, Akt transduces cellular survival signals by inhibiting apoptotic cascades and promoting activation of cell survival pathways. Indeed, previous studies have shown that HUCB cell therapy protects cultured neurons from excitotoxic injury through activation of Akt (Dasari, Veeravalli et al. 2008)

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Because converging lines of evidence demonstrate a role for Akt in the protection of neural cells, the present study examined whether the protection afforded to OLs by HUCB cell therapy occurs through Akt activation. Here we report that HUCB cells rescued OLs and increased the expression of peroxiredoxin 4 (Prdx4) in an Akt dependent manner. Immunohistochemical analysis showed that systemic administration of HUCB cells increased Akt phosphorylation and reduced proteolytic cleavage of caspase 3 following MCAO.

In summary, Akt activation is an integral and necessary transducer of HUCB cell- mediated white matter protection.

Materials and Methods

Animal Care

All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals with a protocol approved by the

Institutional Animal Care and Use Committee at the University of South Florida.

Experiments were designed to minimize the number of animals required.

Sprague-Dawley rats were purchased from Harlan Labs (Indianapolis, IN), maintained on a 12 hr light/dark cycle (6 am – 6 pm) in a climate-controlled room, and allowed access to food and water ad libitum. Neonatal rats birthed from untimed pregnant dams were used for in vitro experiments and 300-350 g male rats were used for in vivo experiments.

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Mixed Glial Culture Preparation

Postnatal day 3 rat pups were decapitated, brains removed, and meninges

dissected away. Rat cortices were dissociated in a solution of 0.25% trypsin/2.21

mM EDTA, triturated, and pelleted. The pellet was re-suspended in DMEM

(dulbecco modified eagle medium) (Mediatech, Manassas, VA) supplemented

with 2.5% fetal bovine serum, 10% horse serum, and 1% antibiotic/antimycotic

(DMEM+). Trypan Blue exclusion was used to assess cell viability. Cells were

seeded (1.5 x 107) into poly-L-lysine-treated 75 cm2 tissue culture flasks. Media

was changed with fresh DMEM+ the following day and cultures were incubated

for 8 days at 37°C (Gottschall, Yu et al. 1995).

OL Cultures Purification

Mixed glial cultures were mechanically shaken for 1 hr to separate

microglial cells from the OL/astrocyte monolayer and media was discarded.

Fresh DMEM+ was added and the flask was returned to the incubator for an

additional 2 days at 37°C. OLs were purified from mixed glial preparations by

shaking the preparations for 18 hrs to separate OLs and microglia from the

astrocyte monolayer. The media was removed, the cells were pelleted and re-

suspended in DMEM+. Viable cells were then counted using Trypan Blue

exclusion. Microglia- and OL-containing media was added to 10 cm plastic tissue culture dishes at a density of 107 cells/dish and incubated for 15 min at

37°C (procedure repeated 3 times for microglial adherence to the plastic). After

incubation, the dishes were gently swirled and media collected. The remaining

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OL suspension was pelleted, re-suspended in DMEM+, and plated on glass poly-

L-lysine-treated coverslips at 3 x 105 cells/coverslip (McCarthy and de Vellis

1980). The following day, media was changed to Neurobasal complete

(Neurobasal supplemented with B-27, L-glutamine 0.5mM, and 10ng/ml PDGF

AA (Platelet derived growth factor AA) (Barres, Schmid et al. 1993; Yang,

Watanabe et al. 2005). OLs remained in Neurobasal complete and PDGF-AA for

7 days to encourage proliferation. After the proliferation period, PDGF-AA was

withdrawn for 5 days to induce OL differentiation into the mature phenotype

(Yang, Watanabe et al. 2005). Experiments were conducted immediately following the 5 day PDGF-AA withdrawal. All in vitro experiments were conducted

using > 95% pure OL cultures, as previously described in Hall et al 2009.

Oxygen Glucose Deprivation

OLs seeded onto glass coverslips were randomly assigned to one of two

conditions: OGD (DMEM without glucose) or normoxia (DMEM with glucose).

Akt inhibitor IV (10µM/ml; EMD4Biosciences, Gibbstown, NJ) was added to

media as treatment required. Transwell inserts (0.2 µm; Nalge Nunc

International, Rochester, NY) were added to 6-well plates containing coverslips.

The inserts provided a barrier that prevented OL-HUCB cell contact but was

permeable to media and soluble factors. Cryopreserved HUCB cells

(ALLCELLS, Emeryville, CA) were rapidly thawed, washed, pelleted to remove

the cryopreservatives and re-suspended in 10 ml DMEM with glucose and

DNase (50 kunitz units/ml; Sigma-Aldrich, St. Louis, MO). HUCB cells were

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seeded onto tissue culture inserts (1x105 cells/insert) and placed into the wells

containing OL coverslips immediately prior to OGD exposure. Experimental

groups not subjected to HUCB cell treatment received inserts containing an

equal volume of DNase-supplemented DMEM with glucose. A negative control

of media alone and wells containing 1x105 HUCB cells with DNase were included

as controls to quantify HUCB cell contribution to the LDH assay for each

experimental condition (Rowe, Leonardo et al.). Akt inhibitor IV was dissolved in

Dimethyl Sulfoxide (DMSO). Akt inhibitor IV was placed in media of groups receiving Akt inhibitor IV treatment at final concentration 10µM/ml. Experimental groups not subjected to Akt inhibitor IV received equivalent quantity of DMSO.

Cells undergoing OGD were placed in an air-tight hypoxia chamber. The chamber was then flushed with hypoxic gas (95% N2, 4% CO2, 1% O2; Airgas,

Tampa, FL) for 15 min and sealed for the duration of exposure. Normoxic cells

were maintained in a standard tissue culture incubator. Cultures were subjected

to OGD or normoxia for 24 hrs at 37°C. The media from each well was collected,

clarified by centrifugation, and lactate dehydrogenase (LDH) analysis was

performed immediately.

LDH Assay

OL cell death in culture was determined using the LDH assay (Takara Bio,

Inc., Madison, WI). Briefly, 100 µl of tissue culture media from each experimental

group was added to a 96-well plate and 100µl of LDH reagent was added to each

well. Plates were incubated for 30 min at 25°C and absorbances were read on a

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microplate reader at a 548 nm wavelength. The media from HUCB cell only cultures served as a control for HUCB cell death. The absorbance of HUCB cell only media, as well as the absorbance of media only, was subtracted from the total absorbance of the OL wells to eliminate background LDH activity. A standard curve was used to quantify OL cell death by extrapolating total numbers of dead OLs from LDH values, as previously described (Rowe, Leonardo et al.).

RNA Collection and Purification

All collection and purification steps were performed under nuclease-free conditions using DNase/RNase-free materials. For RNA lysate collection, 10 µl of

β-Mercaptoethanol (Pharmacia Biotech, Uppsala, Sweden) was added to 1 ml

Mini Kit was used to extract total RNA from each cell lysate using the optional

Qiagen RNase-Free DNase set for DNase digestion (Qiagen Inc, Valencia, CA).

Following the extraction, 1µl of each RNA sample was tested in an Agilent 2100

Bio-analyzer to determine the purity and quantity of RNA present. The remaining sample was stored at -80°C for subsequent use with gene array.

Quantitative Real Time Polymerase Chain Reaction

Primers specific to Prdx4, were purchased from SABiosciences

(Frederick, MD; sequences are proprietary). Total RNA (10 ng/µl) from OL

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cultures were subjected to qRT-PCR. The RT reaction mixture consisted of 3µl

Oligo (dT) Primers, 10 µl cDNA Synthesis Master Mix (2X), 1 µl of Affinity Script

RT/RNase Block enzyme mixture, and RNase-free H2O to a total volume of 20 µl

(Stratagene, La Jolla, CA). The reaction was incubated at 25°C for 5 min to allow

primer annealing, then incubated at 42°C for 45 min to allow cDNA synthesis

followed by 5 min incubation at 95°C to terminate the cDNA synthesis reaction.

Complementary DNA from the RT reaction was added to a PCR reaction mix consisting of 1 µl cDNA, 12.5 µl 2X Brilliant 490 SYBR Green QPCR Master

Mix (Stratagene), 2 µl primer, and nuclease-free PCR grade H2O to a total

volume of 25 µl. The samples were amplified using a BioRad ICycler (Bio-Rad

Laboratories, Hercules, CA) with the following protocol: heating to 95°C for 15

min followed by 40 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 55

°C, and 30 sec of elongation at 72°C. Glyceraldehyde-3-phosphate

dehydrogenase (GADPH) was selected as a reference gene and was used to

calculate the mean normalized expression.

Laser Doppler Blood Flow Measurement

Prior to MCAO surgery, animals were anesthetized with 5% isofluorane/O2

in an induction chamber. Rats were treated prophylactically with ketoprofen (10

mg/kg s.c.), atropine (0.25 mg/kg s.c.) and Baytril (20 mg/kg i.m.) in accordance

with IACUC guidelines. Ketoprofen injections were continued 3 days post-MCAO to minimize pain and discomfort. A constant flow of anesthesia was supplied with an interfaced scavenging system (3-4% isofluorane, flow rate 1 L/min)

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England) Ltd laser Doppler with Moor LAB proprietary Windows-based software.

When surgery was complete, the screw guide was removed, bone wax placed in the burr hole and the scalp incision was sutured. Rats that did not show≥ 60% reduction in blood flow during MCAO were excluded from the study (Hall, Guyer et al. 2009).

Middle Cerebral Artery Occlusion

109

surgeries, the Doppler probe was inserted and the external carotid exposed, but no filament inserted.

HUCB Cells Treatment

To determine whether HUCB cells were protective against stroke-induced injury, rats were injected (i.v, penile vein) with either HUCB cells (1x106 HUCB cells in 500 μL phosphate buffered saline (PBS) (pH 7.4 + DNase) or vehicle

(500 μL PBS + DNase) 48 hrs after MCAO surgery. Sham-operated animals also received vehicle. Animals were then euthanized 54, 72 and 96 hrs post- stroke and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde in PBS. The brains were removed and saturated with 4% paraformaldehyde in PB followed by increasing concentrations of sucrose in PBS

(20%, 30%). Brains were then sectioned at 30 μm on a cryostat to include bregma 1.7mm through bregma -3.3mm, thaw mounted onto slides and stored at

-20°C.

Immunohistochemistry

For peroxidase detection, brain tissue sections/OL coverslips were washed with PBS (pH 7.4) for 5 min and incubated in 3% hydrogen peroxide for

20 min. Sections were then washed 3 times in PBS, incubated for 1 hr in permeabilization buffer (2% serum, 0.3% Triton X-100 and 0.3% 1M lysine in

PBS) and incubated overnight at 4˚C with primary antibody in antibody solution

(2% goat serum, 0.3% Triton X-100 in PBS). The following day, sections were

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washed with PBS and incubated 1 hr at room temperature with secondary

antibody in antibody solution (2% serum, 0.3% Triton X-100 in PBS). Sections

were then washed in PBS, incubated in Avidin-Biotin Complex (ABC; Vector

Laboratories Inc, Burlingame, Ca) mixture for 1hr, washed again and visualized

using a DAB/peroxide solution (Vector Laboratories Inc). After 3 final washes,

sections were dried, dehydrated with increasing concentrations of EtOH (70%,

95%, 100%), cleared with xylene and cover-slipped with DPX mounting medium

(VWR International Ltd, Poole, England). Antibodies consisted of the following:

rabbit anti-Phospho-Akt (Ser473) (1:50; Cell Signaling, Danvers, MA), rabbit anti-

Caspase 3 (1:1000; Sigma-Aldrich, St. Louis, MO). Secondary detection was

achieved using biotinylated secondary antibodies (1:300; Vector Laboratories)

corresponding to the respective species of primary antibodies.

For fluorescent labeling, tissue sections were not incubated in hydrogen

peroxide but otherwise subjected to the same method used for peroxidase

detection, prior to the secondary antibody incubation. Double-label

immunohistochemistry was achieved by co-incubating the tissues with primary

antibodies raised in two distinct species, followed by co-incubation with

secondary antibodies conjugated to distinct fluorophores. Following secondary

antibody incubation, sections were washed and cover-slipped using VectaShield

Hard Set with DAPI (Vector Laboratories). Antibodies used for fluorescent

detection consisted of the following: mouse anti-RIP (1:5000; Millipore,

Temecula, CA), rabbit anti-Phospho-Akt (Ser473) (1:50; Cell Signaling), rabbit anti-Caspase 3 (1:1000; Sigma-Aldrich). Secondary antibodies used were Alexa

111

Fluor 488 and 594 (1:1000; Molecular Probes, Eugene, OR). Negative controls were labeled in the absence of primary antibody corresponding with respective secondary.

Image Analyses

Following in vitro experiments, images were generated using a Zeiss

Axioskop2 microscope controlled by Openlab (Improvision Ltd, Lexington, MA) software. Images were captured with a Zeiss Axiocam Color camera. The

ImageJ 1.410 program (National Institutes of Health, USA) was used to measure total staining intensity and was expressed as ratios of total intensity vs. number of cells, thus showing the relative area positive staining per cell.

Images were captured with a Zeiss Axiocam Color camera. The ImageJ 1.410 program was used to measure total staining intensity and was expressed as percent ratios of ipsilateral vs. contralateral hemispheres. Ratios were calculated for each animal to reduce the influence of edema in the brain hemisphere ipsilateral to the infarct. The groups mean total intensity ratio for each experimental treatment group was used for comparisons across treatments.

Total intensity analysis was conducted where experimenter was blinded of treatment groups.

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Statistical Analyses

Data from all experiments were quantified and analyzed using GraphPad

Prism 4.0 (GraphPad Software, La Jola, CA) software. Main effects were

determined using one-way ANOVAs, followed by Dunnett’s post hoc tests to

detect significant differences across treatment groups. When two variables were

present, two-way ANOVAs were used followed by Bonferroni post hoc tests. A

“p” value < 0.05 was used as the threshold for significant differences.

Results

Akt Inhibition Negated HUCB Cell Protection In Vitro

To assess the role of Akt in HUCB cell protection, Akt inhibitor IV (10

µM/ml) was used to block Akt activation in OL cultures exposed to normoxia or

OGD in the presence or absence of the inhibitor (Figure 1). Exposure to 24 hr

OGD resulted in increased OL cell death as compared to normoxic control (n ≥ 6,

*p < 0.01), whereas HUCB cell co-incubation reduced OGD-induced cell death (n

≥ 6, #p < 0.05). The inhibition of Akt phosphorylation eliminated the protective effects of HUCB cells, as cell death was elevated relative to HUCB only groups and levels were similar to OGD only groups (p > 0.05). Significant differences were not observed in OL cell death among the treatment groups exposed to normoxic conditions (p > 0.05).

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HUCB Cells Increased Akt Phosphorylation In Vitro

Phosphorylation at serine 473 or threonine 308 is essential for Akt-

mediated cell survival (Zhao, Sapolsky et al. 2006). An antibody specific for Akt

phosphorylated at serine 473 was used to evaluate Akt phosphorylation of

cultured OLs (Figure 2). Exposure to 24 hrs OGD increased Akt phosphorylation

compared to normoxic control (n = 3, *p < 0.05). HUCB cell treatment

significantly increased Akt phosphorylation relative to OGD alone (n = 3, #p <

0.05), whereas co-incubation of HUCB cell-treated OLs with Akt Inhibitor IV

reduced phosphorylation to levels detected under normoxic conditions (n = 3 p >

0.05). Akt phosphorylation was not different among treatment groups exposed to

normoxia (Figure 2 E, F, G, H, p > 0.05).

HUCB Cell-Induced Expression of Prdx4 is Suppressed by Akt

Inhibition In Vitro

We have previously reported that HUCB cells increased Prdx4 expression

in OLs exposed to OGD (Rowe, Leonardo et al.). To determine if Prdx4

expression is dependent on Akt activity, qRT-PCR was performed to quantify expression of the Prdx4 gene transcript in OLs subjected to OGD and treated with HUCB cells and/or Akt Inhibitor IV (Figure 3). As expected, HUCB cell treatment increased Prdx4 mRNA when compared to OLs subjected to OGD alone (n ≥ 7, *p < 0.05). Akt inhibiti on reduced Prdx4 mRNA expression both in the presence and absence of HUCB cells (p > 0.05), and these levels were not different from those observed in OLs subjected to OGD alone.

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Delayed HUCB Cell Treatment Increased Akt Phosphorylation

in Cerebral White Matter after MCAO

In vivo Akt phosphorylation was evaluated in the white matter rich region of the external capsule. Rats received HUCB cells or vehicle 48 hrs after MCAO or sham-MCAO and were euthanized at 54, 72 or 96 hrs post-stroke (Figure 4)

for immunochemistry. Constitutive phosphorylation was observed in sham-

operated animals, and levels were not different from vehicle-treated rats at any of

the time points examined. HUCB cell treatment increased Akt phosphorylation at

72 hrs (E) and 96 hrs (H) relative to both vehicle controls and 54 hr HUCB cell-

treated rats (B) (n = 3, # p < 0.05 compared to 54 hr HUCB, * and ^p < 0.05

compared to 72 and 96 hr vehicle, respectively).

HUCB Cells Induce Prdx4 Expression in the External Capsule

Prdx4 expression was assessed to determine whether increased mRNA in

vitro was consistent with increased gene product expression in vivo (Figure 5).

Rats received HUCB cells or vehicle 48 hrs after MCAO or sham-MCAO and

were euthanized at 54, 72 or 96 hrs post-stroke for immunochemistry. Prdx4

immunoreactivity was restricted to cell bodies. In general, sham-operated

controls (Figure 5C,F, I) showed nearly identical staining patterns as vehicle-

treated controls (Figure 5A, D, G) with no apparent differences in Prdx4

expression over the time course examined. Quantification (Figure 5J) showed

that Prdx4 was upregulated in rats treated with HUCB cells compared to sham-

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operated and vehicle-treated controls at 72 hrs post MCAO (*p < 0.01), while no

differences were detected at 54 and 96 hrs.

P-Akt and Caspase 3 Co-localized with OL Marker RIP

Immunofluorescent staining was performed to determine whether

activated caspase 3 (MCAO vehicle-treated rats) (Figure 6 D-F) and phosphorylated Akt (sham-operated rats) (Figure 6 A-C) expression localized to

OLs within the cerebral white matter. Anti-RIP was used to double-label OLs in conjunction with antibodies raised against activated caspase 3 or phosphorylated

Akt. Both proteins were expressed by white matter OLs. Phosphorylated Akt was evident in OL cytoplasm (Figure 6. C), whereas nuclear staining was observed in micrographs depicting caspase 3/RIP co-localization (Figure 6. F).

HUCB Cell Treatment Reduces Caspase 3 Activation in the

External Capsule Following MCAO

The cleavage of caspase zymogens initiates and executes programmed cell death. An antibody generated against activated caspase 3 was used to identify apoptotic signaling in the external capsule of rats following MCAO (Figure

7). As expected, sections from sham-operated controls contained low levels of activated caspase 3. In vehicle-treated animals, levels of activated caspase 3 were elevated at 54 and 72 hrs following MCAO compared to HUCB cell-treated and sham-operated groups (n = 3, *p < 0.05, #p < 0.01 respectively). However, caspase 3 activation returned to basal levels 96 hrs following MCAO in vehicle

116

animals (n = 3, p > 0.05). Additionally, sections from HUCB cell-treated rats

showed no differences in caspase 3 levels compared to sham-operated rats

although caspase 3 positive staining was detected at 54hrs.

Discussion

Oligodendroglias are highly susceptible to ischemic injury (Pantoni, Garcia et al. 1996; Lyons and Kettenmann 1998). In previous studies, soluble factors from HUCB cells protected OLs from hypoxic injury by reducing caspase 3 activation and regulating proliferative, myelin-associated and antioxidant genes in vitro (Rowe, Leonardo et al. ; Hall, Guyer et al. 2009). In vivo experiments have

demonstrated that HUCB cell therapy reduced infarct volume and preserved

cerebral white matter integrity; these effects occurred concomitantly with

increased expression of the antioxidant enzymes Prdx4 and metallothionein 3

(Mt3) (Rowe, Leonardo et al.). These experiments provided strong evidence that

the protection afforded by HUCB cell therapy occurs through the release of

soluble factors that induce antioxidant enzymes to reduce oxidative stress. Here,

data demonstrates that phosphorylation of Akt kinase is one mechanism

responsible for the oligoprotective, antioxidant properties of HUCB cell therapy.

Numerous factors secreted by HUCB cells have been shown to induce Akt

activation, including GDNF, BDNF, LIF, IL-1β, IL-6, IGF1, VEGF and TIMP-1

(Kulik, Klippel et al. 1997; Dolcet, Egea et al. 1999; Jin, Mao et al. 2000; Jin,

Omori et al. 2003; Lee, Yoo et al. 2003; Lentzsch, Chatterjee et al. 2004; Wegiel,

Bjartell et al. 2008). The pro-survival actions of Akt are well known. For example,

117

cellular proliferation and survival have been shown to result from Akt-induced phosphorylation of BAD, the forkhead related family of mammalian transcription factors and glycogen synthase kinase 3β (Zhao, Sapolsky et al. 2006). In the present study, HUCB cells rescued cultured OLs subjected to 24 hrs OGD, whereas the addition of Akt Inhibitor IV completely blocked this effect. Similar results were demonstrated by Dasari et. al., where the ability of HUCB cells to rescue neurons from glutamate excitotoxicity was blocked by Akt inhibition. Thus, the mechanism of oligoprotection observed after HUCB cell treatment is consistent with previous findings and further supports the contention that soluble factors activate Akt signal transduction to increase the viability of cells exposed to ischemic conditions.

We also show that systemic treatment with HUCB cells at 48 hrs post-

MCAO increased Akt activation in the external capsule through phosphorylation at serine 473. These data are in agreement with previous data showing that Akt was rapidly phosphorylated at serine 473 following ischemia in vitro and in vivo, specifically 1 hr following MCAO (Noshita, Lewen et al. 2001; Shibata, Yamawaki et al. 2002; Zhao, Sapolsky et al. 2006). When Akt is unphosphorylated in disease states such as stroke, apoptotic cascades are activated resulting in the release of cytochrome c, caspase 3 and PARP (poly (ADP-ribose) polymerase) cleavage, thus inducing cell death (Nicholson, Ali et al. 1995; Le Rhun, Kirkland et al. 1998; Datta, Brunet et al. 1999; Yu, Andrabi et al. 2006; Zhao, Sapolsky et al. 2006). In the present study, the HUCB cell-induced increase in Akt activation was associated with decreased caspase cleavage, suggesting that HUCB cell

118

treatment attenuated stroke-induced caspase activation through Akt phosphorylation. Furthermore, the lack of phosphorylated Akt in the external capsule of vehicle-treated animals at 96 hrs further supports the notion that unphosphorylated Akt leads to increased cell death through apoptotic signaling.

Indeed, activated caspase 3 was abundant within the cerebral infarct of vehicle- treated rats at this time point.

The formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is also well characterized in ischemic models. These radicals and their associated byproducts produce damage to lipid membranes (Cuzzocrea,

Riley et al. 2001), oxidation, methylation and depurination, producing DNA breaks and mutations that lead to mitochondrial energy failure and subsequent cell death (Steenken 1989; Routledge, Wink et al. 1994; Weitzman, Turk et al.

1994; Cuzzocrea, Riley et al. 2001). Prdx4 is an antioxidant enzyme that detoxifies the cellular environment through peroxidase activity (Hofmann, Hecht et al. 2002; Dewar, Underhill et al. 2003). Here, Akt inhibition arrested HUCB cell-induced Prdx4 mRNA expression in OLs exposed to OGD. Additionally,

Prdx4 protein expression was significantly increased 24 hrs after HUCB cell treatment in the white matter of rats subjected to MCAO, and these elevations correlated with increased phosphorylated Akt.

Conclusions

Previous reports showed that administration of HUCB cells reduced behavioral deficits and infarct volume after stroke and spinal cord injury (Saporta,

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Kim et al. 2003; Vendrame, Cassady et al. 2004; Newcomb, Ajmo et al. 2006;

Hall, Guyer et al. 2009; Chua, Bielecki et al. 2010), yet the precise mechanisms

underlying these protective effects remained unknown. The present study

identified a distinct pathway utilized by HUCB cell therapy to mediate protection.

Data show that Akt phosphorylation is required for oligoprotection and

upregulated expression of the Prdx4 gene transcript in vitro. Furthermore, Akt

phosphorylation is associated with neuroprotection, increased Prdx4 protein

expression and decreased caspase 3 activation in vivo. Taken together, these

data provide strong evidence that the effects of HUCB cells on oligoprotection,

antioxidant enzyme expression and caspase activity converge on Akt signaling.

Future studies investigating specific factors secreted by HUCB cells and the

pathways they activate will be instrumental in establishing more detailed

mechanisms of HUCB cell actions on OLs and the cerebral white matter following

ischemia.

Acknowledgements

This work was supported in part by the National Institutes of Health (R01

NS052839), and the University of South Florida Department of Molecular

Pharmacology and Physiology.

Disclosure

A.E. Willing is a consultant to Saneron CCEL Therapeutics, Inc. and is an inventor on cord blood related patents.

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Figure 1. Akt Inhibition Negates the Protective Effects of HUCB Cells on Cultured OLs. An increase in OL cell death was detected in cultures subjected to 24 hr OGD compared to normoxic controls (n≥ 6, *p < 0.01), demonstrating OGD-induced cellular injury. OL cultures subjected to OGD were rescued by co- incubation with HUCB cells, as cell death was reduced back to levels present in normoxic controls (n ≥ 6, #p < 0.05). Addition of Akt Inhibitor IV eliminated HUCB cells oligoprotective effects in cultures exposed to 24hr ≥ OGD 6). (n Additionally, there was no effect of treatment on OLs exposed to normoxic conditions.

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Figure 2. HUCB Cells Increase Akt Phosphorylation in OLs Subjected to 24hrs OGD. Immunohistochemistry was performed using an antibody generated against Akt phosphorylated at serine 473. Micrographs show immunoreactivity in OLs exposed to OGD (A-D) or normoxia (E-H). Treatment groups were vehicle (A, E), HUCB cells (B, F), Akt Inhibitor IV (C, G) or HUCB cells + Akt Inhibitor IV (D, H). Quantification (I) revealed increased Akt phosphorylation in OLs exposed to OGD relative to normoxic controls (n = 3, *p < 0.05). Addition of HUCB cells resulted in an additional increase in phosphorylation during OGD relative to vehicle-treated OGD controls (n = 3, #p < 0.05). Akt Inhibitor IV reduced phosphorylation during OGD when added in the presence or absence of HUCB cells, such that immunoreactivity was not different from normoxic controls. Scale bars = 50 μm. 128

Figure 3. Akt Inhibitor IV Suppresses HUCB Cell Induced Prdx4 Expression During OGD. qRT-PCR was performed to quantify mRNA expression of the antioxidant enzyme Prdx4. OLs were subjected to OGD and treated with HUCB cells in the presence or absence of Akt Inhibitor IV. HUCB cells increased Prdx4 gene transcript compared to vehicle-treated controls (n ≥ 7, *p < 0.05,). Co - incubation of the Akt inhibitor with HUCB cells reduced the HUCB cell-induced elevation in Prdx4 mRNA back to levels detected in OLs treated with vehicle or Akt inhibitor alone.

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Figure 4. HUCB Cells Increase Akt Phosphorylation In Vivo. Immunohistochemistry was performed using an antibody generated against Akt phosphorylated at serine 473. Micrographs show immunoreactive cells (arrows) in the ipsilateral external capsule of vehicle-treated (A, D, G), HUCB cell-treated (B, E, H) and sham-operated (C, F, I) rats. These rats were euthanized at 54 (A- C), 72 (D-F) or 96 hrs (G-I) after MCAO. Quantification (J) revealed increased phosphorylation of Akt in tissues from HUCB cell-treated rats euthanized at 72 (^p < 0.05) and 96 hrs (*p < 0.05) compared to vehicle-treated rats at the respective time points. Additionally, phosphorylated Akt was increased after HUCB cell treatment at 72 hrs relative to 54 hrs (n = 3, #p < 0.05), while no differences were detected between groups at the 54 hr time point. Scale bars = 50 μm.

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Figure 5. HUCB Cells Induce Prdx4 Expression In Vivo. Immunohistochemistry was performed to determine the effects of HUCB cell therapy on Prdx4 protein expression. Micrographs show immunoreactive cells (arrows) in the ipsilateral external capsule of vehicle-treated (A, D, G), HUCB cell-treated (B, E, H) and sham-operated (C, F, I) rats. These rats were euthanized at 54 (A-C), 72 (D-F) or 96 hrs (G-I) after MCAO. Quantification (J) revealed increased expression of Prdx4 in tissues from rats treated with HUCB cells and euthanized at 72 hrs post-MCAO relative to vehicle-treated and sham- operated animals at the same time point (n=3. *p < 0.01). No differences were detected across treatment groups at any other time points examined. Scale bars = 50 μm.

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Figure 6. Akt and Caspase 3 are Activated in OLs In Vivo. Confocal micrographs show immunoflourescent co-localization using the OL specific antibody anti-RIP (A, D) in combination with antibodies generated against phosphorylated Akt (B, MCAO sham-operated vehicle treated rat) or activated caspase 3 (E, MCAO vehicle-treated rat) in the external capsule. Merged images demonstrate expression of phosphorylated Akt (C) and activated caspase 3 (F) in RIP-positive OLs. Arrows denote immunoreactivity. Scale bars = 50 μm.

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Figure 7. HUCB Cells Reduce Caspase 3 Activation In Vivo. Immunohistochemistry was performed to determine the effects of HUCB cell therapy on caspase 3 activation. Micrographs show immunoreactive cells (arrows) in the ipsilateral external capsule of vehicle-treated (A, D, G), HUCB cell-treated (B, E, H) and sham-operated (C, F, I) rats. These rats were euthanized at 54 (A-C), 72 (D-F) or 96 hrs (G-I) after MCAO. MCAO induced caspase 3 activation was detected 54 (A) and 72 hrs (D) post MCAO. Quantification (J) revealed HUCB cell treatment blocked the stroke-induced activation of caspase 3 detected in vehicle-treated rats at 54 and 72 hrs (n = 3, *#p < 0.05) such that levels were not different from sham-operated controls. No differences were detected across groups at the 96 hr time point. Scale bars = 50 μm.

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CHAPTER FOUR

LEUKEMIA INHIBITORY FACTOR AND GRANULOCYTE COLONY

STIMULATING FACTOR INDUCE ANTIOXIDANTS TO ENHANCE SURVIVAL

OF OLIGODENDROCYTES EXPOSED TO OXYGEN GLUCOSE

DEPRIVATION

D.D. Rowe, MS, J Recio, MS, L.A. Collier, BS, A.E. Willing, PhD,

K.R. Pennypacker, PhD.

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Abstract

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with numerous

effects on cellular survival and function. Human umbilical cord blood (HUCB)

cells secrete soluble factors to protect neurons and oligodendrocytes (OL)s from

oxygen glucose deprivation (OGD). LIF is a soluble factor expressed and

secreted by HUCB cells. We investigated the effect of LIF on OLs exposed to 24

hrs oxygen glucose deprivation (OGD). Administration of LIF attenuated OGD

induced OL cell death by increasing expression of antioxidants metallothionein

(Mt3) and peroxiredoxin (Prdx4). The addition of an Akt inhibitor (Akt inhibitor IV) abolished LIF protective effects and blocked LIF induced Prdx4 expression.

Granulocyte colony stimulating factor (GCSF) reduced OL cell death, an effect that was blocked by Akt inhibitor. Additionally LIF/GCSF treatment did not reduce cell death appreciable from LIF treatment alone. We have examined two soluble factors secreted by HUCB cells and have shown that the optimization of each monotherapy is just as efficacious as HUCB cell treatment in vitro.

Introduction

Following ischemic injury, the white matter alike the gray matter is

adversely affected by reduced cerebral blood flow. Neuronal death attributed to

ischemia has been extensively studied, but our knowledge of white matter

pathology following an ischemic insult remains limited. Oligodendrocytes (OL)s

predominate the white matter tract and of the glial cells are most susceptible to

ischemic injury (Lyons and Kettenmann 1998). This susceptibility has been linked

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to the inherently high metabolic rate, iron content and reduced antioxidant

production capacity of OLs (Braughler, Duncan et al. 1986; Connor and Menzies

1996; Juurlink 1997; Juurlink, Thorburne et al. 1998).

Cell based therapy has shown promise in stroke research, specifically

human umbilical cord blood (HUCB) cells. HUCB cells migrate to the infarct area,

reverse stroke induced pathology by rescuing neurons, glial cells and enhancing behavioral recovery post stroke (Vendrame, Cassady et al. 2004; Vendrame,

Gemma et al. 2005; Hall, Guyer et al. 2009; Rowe, Leonardo et al. 2010). HUCB cells attenuate stroke pathology in the white matter tract by halting the activation and progression of apoptotic cascades and, increasing the expression of survival associated proteins in OLs (Rowe, Leonardo et al. 2010). Although promising, cell based therapies are difficult to standardize. Therefore, it is important to understand the mechanism by which these cells mediate cellular survival and behavioral recovery post stroke. HUCB cells secrete soluble factors to protect OL exposed to oxygen glucose deprivation (Rowe, Leonardo et al. 2010). A number of these soluble factors have been identified as cytokines, chemokines, metalloproteinase inhibitors, growth factors and interleukins (Neuhoff, Moers et al. 2007).

Leukemia inhibitory factor (LIF) is a 180 amino acid single 4-α-helix glycoprotein that binds to the LIF cell surface receptor complex which includes the LIF receptor β (LIFR) and the gp130 receptor chain (Kurek 2000; Metcalf

2003). LIF rescued OLs and reduced demyelination following spinal cord injury through STAT 3 and PI3/Akt survival pathway activation (Metcalf and Gearing

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1989; Azari, Profyris et al. 2006). Furthermore intravenous administration of LIF

attenuated demyelination and OL loss in experimental autoimmune

encephalomyelitis (EAE), the mouse model of multiple sclerosis, the addition of

antibodies generated against LIF exacerbated EAE disease pathology (Metcalf

and Gearing 1989; Metcalf and Gearing 1989; Butzkueven, Zhang et al. 2002). In ischemic conditions, increased LIF was detected in neuronal cell bodies and axons of rats subjected to MCAO. Where in cultures, LIF expression was upregulated in astrocytes exposed to OGD (Slevin, Krupinski et al. 2008).

Furthermore, intracerebral injections of LIF following focal ischemia attenuated ischemic brain injury (Suzuki, Yamashita et al. 2005). Specifically in culture, LIF administration rescued mature OLs from the pro inflammatory cytokines interferon γ and tumor necrosis factor α induced cell death, through the activation of both JAK/STAT and PI3/Akt pathway (Slaets, Dumont et al. 2008).

GCSF is a 19.6 kDa glycoprotein that alike LIF and other HUCB cell secreted factors activate the Akt pathway (Nagata, Tsuchiya et al. 1986; Hunter and Avalos 1998; Neuhoff, Moers et al. 2007; Slaets, Dumont et al. 2008).

Specifically in stroke, GCSF has been shown to be neuroprotective (Solaroglu,

Cahill et al. 2006). Following MCAO, GCSF administration reduced the inflammatory response to stroke. This study highlighted the down regulation of nuclear factor kappa B (NFκB) and inducible nitric oxide synthase (iNOS) in the infarct area (Sehara, Hayashi et al. 2007). Additionally, in a small study conducted in 2006 GCSF was proven safe and effective at improving neurologic outcomes in patients who experienced strokes affecting the MCA (Shyu, Lin et al.

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2006). Furthermore, a more recent study (Ax 200) has been completed and the

results are being prepared for publication.

In this study, we examined whether LIF can duplicate HUCB cell protective effects. We report that LIF attenuated OGD induced OL cell death in vitro where the addition of Akt inhibitor IV blocked LIF protection. As with HUCB cell experiments, administration of LIF upregulates antioxidants Prdx4 and Mt3 resulting in reduced oxidative stress. Examining another soluble factor secreted by HUCB cells, administration of granulocyte colony stimulating factor (GCSF) rescued OL subjected to 24 hrs OGD and again Akt inhibitor IV blocked GCSF effects. Thus, HUCB cells release at least two soluble factors that promote oligoprotection via Akt and antioxidant enzyme activity.

Materials and Methods

Animal Care

All animal procedures were conducted in accordance with the NIH Guide

for the Care and Use of Laboratory Animals with a protocol approved by the

Institutional Animal Care and Use Committee at the University of South Florida.

Experiments were designed to minimize the number of animals required.

Sprague-Dawley rats were purchased from Harlan Labs (Indianapolis, IN),

maintained on a 12 hrs light/dark cycle (7 am – 7 pm) in a climate-controlled

room, and allowed access to food and water ad libitum. Neonatal rats birthed

from untimed-pregnant dams were used for in vitro experiments and 300-350 g

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male rats were used for in vivo experiments. Measures taken to minimize pain and discomfort are described in the subsequent methodology.

Mixed Glial Culture Preparation

DMEM+ the following day and cultures were incubated for 8 days at 37°C

(Gottschall, Yu et al. 1995).

OL Cultures Purification

Mixed glial cultures were mechanically shaken for 1 hr to separate microglial cells from the OL/astrocyte monolayer and media was discarded.

Fresh DMEM+ was added and the flask was returned to the incubator for an additional 2 days at 37°C. OLs were purified from mixed glial preparations by shaking the preparations for 18 hrs to separate OLs and microglia from the astrocyte monolayer. The media was removed, the cells were pelleted and re- suspended in DMEM+. Viable cells were then counted using Trypan Blue exclusion. Microglia- and OL-containing media was added to 10 cm plastic

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tissue culture dishes at a density of 107 cells/dish and incubated for 15 min at

37°C (procedure repeated 3 times for microglial adherence to the plastic). After incubation, the dishes were gently swirled and media collected. The remaining suspension was pelleted, re-suspended in DMEM+, and plated on glass poly-L- lysine-treated coverslips at 3 x 105 cells/coverslip (McCarthy and de Vellis 1980).

The following day, media was changed to Neurobasal complete (Neurobasal supplemented with B-27, L-glutamine 0.5mM, and 10ng/ml PDGF AA) (Barres,

Schmid et al. 1993; Yang, Watanabe et al. 2005). OLs remained in Neurobasal complete and PDGF-AA for 7 days to encourage proliferation. After the proliferation period, PDGF-AA was withdrawn for 5 days to induce OL differentiation into the mature phenotype (Yang, Watanabe et al. 2005).

Experiments were conducted immediately following the 5 day PDGF-AA withdrawal. All in vitro experiments were conducted using > 95% pure OL cultures, as previously described in (Hall, Guyer et al. 2009).

Oxygen Glucose Deprivation

OLs were seeded onto glass coverslips and randomly assigned to one of two conditions: OGD (DMEM without glucose) or normoxia (DMEM with glucose). LIF/GCSF (Millipore, Billerica, MA: 10-1000ng/ml) was added to media as treatment required. Akt inhibitor IV (EMD4Biosciences, Gibbstown, NJ:

10µM/ml) was added to media as treatment required. Experimental groups not subjected to LIF treatment an equal volume of buffer which LIF was dissolved in

(50mM sodium phosphate/1mMDTT/10% glycerol/250mM Nacl, pH 7.4 and 140

0.02% Tween 20) supplemented DMEM. A negative control of media alone and

wells containing LIF or GCSF were included as controls to quantify media and

buffer solution contribution to LDH assay for each experimental condition. Akt

inhibitor IV was dissolved in DMSO. Akt inhibitor IV was placed in appropriate

media at final concentration 10µM/ml. DMSO was added to medium in

experimental groups not subjected to Akt inhibitor IV.

For treatment with HUCB cells, Transwell inserts (0.2 µm; Nalge Nunc

International, Rochester, NY) were added to 6-well plates containing coverslips.

The inserts provided a barrier that prevented OL-HUCB cell contact but are